Electrophysiological assays using oocytes that express human enac and the use of phenamil to improve the effect of enac enhancers in assays using membrane potential reporting dyes

ABSTRACT

In one aspect, the present invention relates to a mammalian cell-based high-throughput assay for the profiling and screening of human epithelial sodium channel (hENaC) cloned from a human kidney c-DNA library and is also expressed in other tissues including human taste tissue. The present invention further relates to amphibian oocyte-based medium-throughput electrophysiological assays for identifying human ENaC modulators, preferably ENaC enhancers. Compounds that modulate ENaC function in a cell-based ENaC assay are expected to affect salty taste in humans. The assays described herein have advantages over existing cellular expression systems. In the case of mammalian cells, such assays can be run in standard 96 or 384 well culture plates in high-throughput mode with enhanced assay results being achieved by the use of a compound that inhibits ENaC function, preferably an amiloride derivative such as Phenamil. In the case of the inventive oocyte electrophysiological assays (two-electrode voltage-clamp technique), these assays facilitate the identification of compounds which specifically modulate human ENaC. The assays of the invention provide a robust screen useful to detect compounds that facilitate (enhance) or inhibit hENaC function. Compounds that enhance or block human ENaC channel activity should thereby-modulate salty taste in humans.

This application is a divisional of U.S. application Ser. No.15/257,517, filed Sep. 6, 2016, which is a divisional of U.S. Utilityapplication Ser. No. 10/563,758 filed Mar. 6, 2008, now U.S. Pat. No.9,459,259, which is a 371 of International PCT Application No.PCT/US04/21853 filed Jul. 9, 2004, which claims priority from U.S.Provisional Application Ser. No. 60/485,745 filed Jul. 10, 2003, USProvisional Application Ser. No. 60/287,413, filed May 1, 2001 and is acontinuation-in-part of U.S. Utility application Ser. No. 10/133,573filed Apr. 29, 2002, all of which are incorporated herein by referencein their entireties.

SEQUENCE DISCLOSURE

This application includes as part of its disclosure a sequence listingtext file named “43268o2006.txt” which was created on Jan. 10, 2019 andhas a size of 34,831 bytes, which is hereby incorporated by reference inits entirety.

The present invention involves the discovery that the efficacy ofcell-based assays that screen for compounds that modulate ENaC function,preferably ENaC enhancers, is improved by the further use of a compoundthat at least partially inhibits ENaC function preferably an amiloridederivative such as phenamil. The present invention further relates toimproved electrophysiological assays that identify human ENaC modulatorsusing oocytes, preferably frog oocytes, that express a functional humanENaC sodium channel.

FIELD OF THE INVENTION

The present invention in part relates to novel cell based assays thatuse recombinant host cells that express amiloride-sensitive sodiumchannels to profile, screen for, and identify taste modulatingcompounds. More specifically, the invention relates to assays thatutilize test cells that express a functional human epithelial sodiumchannel (hENaC), preferably amphibian oocytes or mammalian cells, andthe use of these test cells in cells in high throughout or moderatethroughput cell-based assays, preferably electrophysiological assays, toidentify compounds that enhance or block hENaC function.

BACKGROUND OF THE INVENTION

An amiloride-sensitive epithelial sodium channel (ENaC) mediates sodiuminflux across the apical membrane of taste buds cells in the tongue(Heck, et al, Science (1984) 223: 403-405). ENaC, a member of theENaC/degenerin superfamily of ion channels involved in sodium transport,is composed of three partially homologous α, β, and γ subunits expressedat both the RNA and protein level in fungiform, foliate, andcircumvallate papilla as well as the lingual epithelium in taste tissue(Li, et al, Proc. Natl. Acad. Sci. (1994) 91: 1814-1818; Kretz, et al,J. Histochem. Cytochem. (1999) 47(1): 51-64; Lin, et al, J. Comp.Neurol. (1999) 405: 406-420; Xiao-Jiang, et al, Mol. Pharmacol. (1995)47: 1133-1140).

Complementary DNAs (cDNAs) encoding amiloride-sensitive epithelialsodium channel (ENaC) channel subunits have been isolated from kidneycells and expressed in a mammalian cell line. The channel expressed inthis system has been shown to have similar properties to the distalrenal sodium channel, i.e., high sodium selectivity, low conductance,and amiloride sensitivity. One form of the naturally occurring ENaCchannel is comprised of three subunits of similar structure: alpha (OMIMEntry 600228), beta (OMIM Entry 600760), and gamma (OMIM Entry 600761).Each of the subunits is predicted to contain 2 transmembrane spanningdomains, intracellular amino- and carboxy-termini, and a cysteine-richextracellular domain. The three subunits share 32 to 37% identity inamino acid, sequence. Alternatively spliced forms of alpha-ENaC havealso been identified, indicating heterogeneity of alpha subunits ofamiloride-sensitive sodium channels that may account for the multiplespecies of proteins observed during purification of the channel (SeeU.S. Pat. No. 5,693,756, which is herein incorporated by reference).

An inhibitor of ENaC sodium channel function, amiloride, is known toattenuate gustatory responses to sodium chloride in numerousnon-mammalian as well as mammalian species, including humans (Halpern,Neuroscience and Behavior Reviews (1998) 23: 5-47 and all referencescited within; Liu, et al, Neuron (2003) 39: 133-146; Zhao, et al, Cell(2003) 115: 255-266). In humans, amiloride reportedly reduces theintensity of sodium chloride by 15-20% when used at concentrations thatspecifically inhibit ENaC function (Halpern, Neurosciences and BehaviorReviews (1998) 23:5-47 and all references cited within; Feldman, et al,J. Neurophysiol. (2003) 90(3): 2060-2064). Therefore, compounds thatincrease the transport of sodium ions through ENaC channels may functionas general salt taste enhancers and augment human salt taste perceptionas suggested in our previous patent application (PCT WO 02/087306 A2).Further, based on published electrophysiological data and the discoverythat a human ENaC is expressed in taste bud cells, a model of saltytaste transduction mediated by ENaC has been constructed. As such, theuse of ENaC in the identification of substances which stimulate or blocksalty taste perception has been suggested (See U.S. Pat. No. 5,693,756,supra and PCT WO 02/087306 A2).

Cell-based functional expression systems commonly used for thephysiological characterization of ENaC are Xenopus laevis oocytes andcultured mammalian cell lines. The oocyte system has advantages overmammalian cells in that it allows the direct injection of multiplemRNAs, provides high levels of protein, expression, and can accommodatethe deleterious effects inherent in the over expression of ENaC.However, the drawbacks of this system are that electrophysiologicalrecording in Xenopus oocytes is not amenable to screening large numbersof compounds and the Xeropus oocyte is an amphibian not a mammaliansystem. Studies of the electrophysiological properties of rodent ENaC inmammalian cell lines (HEK293 and M DCK) stably expressing the channelhave been reported in the literature. While these studies used mammaliancell lines, channel function was assayed using tediouselectrophysiological techniques. Such approaches do not lend themselvesto high throughput screening of compounds. Thus, there remains a need inthe art for identification of salt taste enhancers amenable to highthroughput screening.

The development of salt taste enhancers has been the focus of numerousprior scientific publications and patents. However, direct modulation ofthe ENaC sodium channel involved in salt taste perception is a novel andunique approach to enhance human salty taste. Some examples ofpreviously reported salt enhancing compounds and their properties arediscussed below.

Some proteolzyed proteins, peptides, amino acids, and amino-acid estersreportedly function as salt enhancers (Tamura, et al, Argic. Biol. Chem.(1989) 53(6): 1625-1633, 1989; U.S. Pat. No. 5,711,985). However, theseagents require high concentrations, between 30-60 mM, and must besupplemented with hydrochloride acid to positively modulate salty taste.In addition, the cost and difficulty in synthesizing these compounds areprohibitive for their large-scale commercial use as salt enhancers forthe general population.

Choline chloride, an ammonium salt classified by the federal governmentas a GRAS (generally regarded as safe) compound, has been reported tofunction as a salt enhancer in humans and rodents. In humans, cholinechloride increases the saltiness of dilute salt solutions (less than 50mM NaCl) by a factor of two and reportedly increases the preference orhedonic ratio of both cooked peas and Campbell's low salt tomato soup(Locke, et al, Physiology & Behavior (1994) 55(6): 1039-1046; U.S. Pat.Nos. 5,260,091; 5,260,049). However, similar to peptides and amino acidsdescribed above, choline chloride requires significant concentrations(in the mM range) to enhance salty taste.

Derivatives of amiloride, which do not block ENaC function but insteadblock sodium-proton exchange, as well as chloride channel blockers, suchas IAA-94 and anthranilic acid, reportedly increase fluid intake; anindirect measurement of salt consumption, in a rodent model system (U.S.Pat. No. 5,260,091). However, the utility of these agents as human saltenhancers has not been reported.

Cetylpyridiunium chloride (CPC) has been reported to increaseamiloride-insensitive nerve responses to salt in rats and to enhance thesaltiness of low salt Campbell's tomato soup by 50% in humans when usedat low concentrations (high uM range) (DeSimone, et al. J. Neurphysiol.(2001) 86: 2638-2641; U.S. Pat. No. 4,997,672). However, CPC is adetergent and based on its structure likely intercalates into lipidbilayers of cells' and thereby non-specifically activates salt tastecells by disrupting lipid homeostasis. Indeed, high concentrations ofCPC (low mM range), above the critical micelle concentration, actuallyinhibit rat nerve responses to numerous salty compounds including sodiumchloride, potassium chloride, and ammonium chloride, furthersubstantiating that the reportedly observed CPC effects were likelynon-specific.

Trehalose, a disaccharide composed of two glucose molecules, reportedlyincreases the saltiness of sodium chloride solutions by 1.2 to 2-fold(U.S. Pat. No. 6,159,529). Similar to peptides and choline chloride,high levels (1.5-12%) of this sugar are required to enhance saltiness,suggesting that the observed effects could be non-specific andattributable to taste cell volume changes (cell shrinkage) due tohyperosmolarity. In addition, the specificity of trehalose and otheraforementioned salt enhancers to enhance salty taste and not modulateother tastes, including sweet, bitter, sour, and umami, was notaddressed.

Alapyridaine, a derivative of the amino acid alanine that is formed as aby-product in heated sugar/amino acid mixtures, reportedly decreases thethreshold for detecting sodium chloride 5-fold (Soldo, et al, ChemicalSenses (2003) 28: 371-379, 2003; Ottinger, et al, J. Agric Food Chem(2003) 51: 1035-1041, 2003). Alapyridaine, however, reportedly functionsas a general taste enhancer and decreases the detection thresholds forsalt as well as sweet and umami tastes. In addition, the effect ofalapyridaine on salt taste at higher, more physiologically-relevant,salt concentrations was not disclosed. Thus, the effects of alapyridainemay only surface when tasting low salt concentrations near thresholddetection-levels.

The antibiotic novobiocin also reportedly enhances nerve responses tosodium chloride in rats (Feigin, et al. Am. J. Physiol. (1994) 266:C1165-C1172). However, disadvantageously novobiocin reportedly formsamiloride-insensitive cation-selective ion channels in lipid bilayerssuggesting that this agent pokes holes in cell membranes and, perhapssimilar to CPC, non-specifically increase taste cell activity. Theeffect of novobiocin on human salt taste perception has not beenreported.

Bretylium tosylate, an anti-fibrillary drug that modulates adrenergicand muscarinic receptors, has been reported to specifically potentiatesalt taste in rodents and humans without affecting sweet, sour, orbitter taste (Schiffman, et al, Physiology & Behavior (1986) 36:1129-1137). However, a significant disadvantage of bretylium tosylate,separate from the relatively high concentrations required to positivelymodulate salty taste (mM range), is that the compound is a therapeuticused to treat cardiac patients. Consequently this compound would beunsuitable for use in the general population.

Glybenclamide, an inhibitor of members of the ATP-binding cassette (ABC)protein superfamily, including the cystic fibrosis transmembraneconductance regulator and the sufonylurea receptor, reportedly increasesamiloride-sensitive ENaC sodium current by doubling the open probabilityof individual ENaC channels (Chraibi, et al. The Journal of Pharmacologyand Experimental Therapeutics (1999) 290: 341-347, 1999; Schnizler, etal, Biochemica et Biophysica Acta (2003) 1609: 170-176). However,because Glybenclamide modulates ABC protein function, it is probablethat Glybenclamide effects are due to indirect modulation of ENaCactivity by ABC proteins and not attributable to direct modulation ofENaC channel function. In addition, glybenclamide has not beendemonstrated to enhance human salt taste perception nor hasglybenclamide been suggested as a salt taste enhancer.

Thus, based on the foregoing, it is evident that improved methods foridentifying compounds that specifically modulate ENaC and salty tasteare needed as are improved salty taste modulators. Preferably, suchmethods will comprise high or medium throughput methods and will screenfor compounds having a direct effect on human ENaC function.

Summary of the Invention

The present invention obviates the problems of the prior art, relatingto assays for identifying compounds that modulate ENaC. Specifically,the present invention provides cell-based assays that utilizerecombinant host cells, preferably mammalian cells or oocytes thatexpress a functional human ENaC to identify compounds that modulate ENaCand consequently salty taste. More specifically, the present inventionprovides oocyte and mammalian cell-based assays, preferably high ormedium throughput, for the profiling and screening of a sodium channel,more particularly an amiloride-sensitive epithelial sodium channel(ENaC), which assays optionally may include the addition of a compoundthat partially or totally inhibits ENaC function, preferably amilorideor an amiloride derivative such as phenamil. It has been found that theuse of phenamil in particular enhances signal intensity during assays,preferably high or medium throughput assays for identifying compoundsthat modulate (enhance or inhibit) ENaC function. Such methods can beused to functionally characterize ENaC activity or to identify compoundsthat either enhance or block salty taste perception (herein referred toas salty taste modulators).

Accordingly, in a first aspect the invention provides recombinant hostcells, preferably mammalian cells or amphibian oocytes that express afunctional hENaC. In a preferred embodiment these cells will transientlyor stably express all three subunits of hENaC (alpha or delta, beta andgamma), or transiently or stably express one or more subunits orfunctional chimeras, variants or fragments thereof. Mammalian cellssuitable for use in the invention encompass any mammalian cell capableof expressing a functional hENaC, including by way of example COS, CHO,MDCK, HEK293, HEK293T, NIH3T3, Swiss3T3 and BHK cells. However, in thepreferred embodiment the invention provides HEK293T cells that express afunctional hENaC. Oocytes useful in the invention preferably includeamphibian oocytes, e.g., Xenopus oocytes.

In a second aspect, the invention provides cell-based assays thatutilize mammalian cells or amphibian oocytes that express a functionalENaC, preferably hENaC, to identify compounds, including e.g., smallorganic molecules, antibodies, peptides, cyclic peptides, lipids andnucleic acids that enhance or block ENaC function. Preferably, theseassays w ill include the addition of known ENaC inhibitor at aconcentration that partially inhibits ENaC function prior to addition ofuse of one or more putative ENaC modulatory compounds.

Preferably the assay will comprise a mammalian or oocyte cell-basedassay, preferably high or medium throughput, for the profiling andscreening of putative modulators of an epithelial sodium channel (ENaC)comprising: (i) contacting a test cell expressing an ENaC loaded with amembrane potential fluorescent dye or a sodium-sensitive fluorescent dyewith at least one putative modulator compound in the presence of abuffer containing sodium; (ii) prior to the addition of said at leastone putative modulator compound, contacting said host cell with acompound that is known to inhibit ENaC function, at a concentrationwhereby ENaC function is at least partially inhibited, preferably anamiloride derivative such as phenamil; and (iii) monitoring changes influorescence of the membrane potential dye or sodium-sensitive dye incells contacted with the putative modulator plus sodium after additionof the known ENaC inhibitor compound compared to the change influorescence of the membrane potential dye or sodium-sensitive dye forcells contacted with sodium alone to determine the extent of ENaCmodulation.

In another preferred aspect of the invention, a method for monitoringthe activity of an epithelial sodium channel (ENaC) is providedcomprising: (i) providing test cells, e.g., mammalian cells transfectedor transformed with a functional ENaC; (ii) seeding the test cell in thewell of a multi-well plate and incubating for a time sufficient to reachat least about 70% confluence; (iii) dye-loading the seeded test cellswith a membrane potential fluorescent dye or sodium-sensitivefluorescent dye in the well of the multi-well plate; (iv) contacting thedye-loaded test cell with at least one putative modulatory compound inthe well of the multi-well plate; (v) prior to the addition of said atleast one putative modulatory compound, further contacting said hostcell with a compound that partially inhibits ENaC function, e.g., anamiloride derivative such as phenamil; and (vi) monitoring any changesin fluorescence using a fluorescence plate reader.

In another preferred embodiment of the invention (i) suitable cells,e.g., HEK293T cells or another mammalian cell line are transformed,transfected with DNA sequences encoding subunits necessary to produce afunctional human ENaC; (ii) the cells are seeded onto a multi-wellplates, e.g., 384 well plates, preferably to about 80% confluence; (iii)the seeded test cells are loaded with a membrane potential sensitive dyesuch as CC2-DMPVE or DiSBAC2(3); (iv) the dye-loaded cells are thencontacted with at least one putative ENaC modulatory compound; (v) thedye-loaded cells are preferably further contacted prior to contactingwith said at least one ENaC modulatory compound with an amount of atleast one known ENaC inhibitor at a concentration that results in atleast partial ENaC inhibition: and (iv) monitoring changes in cellfluorescence using a voltage intensity plate reader e.g., VIPRII (AuroraBiosciences).

In yet another aspect of the invention, a method for identifying a saltytaste modulatory compound is provided comprising: (i) providing testcells transfected, transformed with a functional human ENaC; (ii)seeding the test cell in the well of a multi-well plate and incubatingfor a time sufficient to reach at least about 70% confluence morepreferably to about 80%, confluence; (iii) dye-loading the seeded testcells with a membrane potential dye in the well of the multi-well plate;(iv) contacting the dye-loaded test cells with at least one putativemodulatory compound in the well of the multi-well plate; (v) preferablyprior to the addition of said at least one putative modulatory compoundfurther contacting the dye-loaded test cell with a known ENaC inhibitorcompound at a concentration that at least partially inhibit ENaCfunction, e.g., an amiloride derivative such as phenamil; (vi)monitoring any changes in fluorescence of the membrane potential dye dueto modulator/ENaC interactions using a fluorescence plate reader; and(vii) identifying the at least one putative modulator as a salty tastemodulating compound based on the monitored changes in fluorescence.

In yet another preferred embodiment of the invention (i) suitable cells,e.g., HEK293T cells are transformed or transfected with DNA sequencesencoding subunits necessary to produce a functional human ENaC; (ii) thecells are seeded onto multi-well plates, e.g., 384 well plates,preferably to about 80% confluence; (iii) the seeded test cells areloaded with a membrane potential sensitive dye such as CC2-DMPVE orDiSBAC2(3); (iv) the dye-loaded cells are then contacted with at leastone putative ENaC modulatory compound; (v) preferably prior thereto thedye-loaded cells are contacted with a compound known to inhibit ENaCfunction, e.g., an amiloride derivative such as phenamil at aconcentration that at least partially inhibits ENaC function; (vi)changes in cell fluorescence are monitored using a voltage intensityplate reader e.g., VIPRII (Aurora Biosciences); and (vii) compounds thatmodulate salty taste are selected based on a change in fluorescenceintensity.

In yet another preferred embodiment of the invention,electrophysiological assays, preferably two-electrode voltage clampassays are provided wherein human ENaC modulatory compounds areidentified based on their effect (inhibitory or enhancing) onmacroscopic electrical current in oocytes, preferably amphibian oocytes(frog)), that express a functional human EnaC sodium channel. Theseassays are an improvement over some other cell-based assays foridentifying ENaC modulators, in part because oocytes express fewendogenous ion channels; consequently the oocyte expression systemadvantageously allows direct measurement of ENaC sodium channel currentwith little or no background.

In another preferred embodiment of the invention, theseelectrophysiological assays will further contact said oocytes with atleast a partial inhibitor of ENaC, e.g., amiloride or an amiloridederivative as a control or in order to enhance measurable changes insodium channel cell current.

In yet another preferred embodiment of the invention, frog oocytes thatfunctionally express a human ENaC sodium channel are provided whichexpress human ENaC alpha, beta and gamma or delta, beta and gammasubunits.

In another preferred embodiment of the invention, the ENaC used incell-based assays according to the invention can be composed ofnaturally occurring human ENaC subunits, one or more alternativelyspliced human ENaC subunits, or a functional variant thereof.Alternatively, the ENaC can be composed of at least the alpha subunit ofa naturally occurring human ENaC, or a n alternatively spliced versionthereof. In another embodiment, a delta subunit (such as Genebankaccession U38254; see J Biol. Chem, 270(46):27411-4 (1995)) or a variantthereof can substitute for the alpha subunit.

Preferably, these subunits are encoded by SEQ ID: NO.: 1, 2, 3 and 7disclosed infra. These and other aspects of the invention will becomeapparent to one of skill in the art from the following detaileddescription, drawings, and claims.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the functional expression of hENaC resulting in asodium dependent amiloride sensitive fluorescence change. Transaction ofHEK293T cells with varying 1:1:1 ratios of α, β, and γ, subunit plasmidsof human kidney ENaC results in a Na⁺ dependent amiloride sensitivevoltage change, as compared to mock transfected cells. A, B, C, and Dwere transfected with 111:1 rations of α, β, and γ plasmid at absolutelevels of 4.4.1. and 0.25 respectively. E and F were mock transfectedwith Beta-gal and pUC. Transfection efficiency was approximately 40% andcell density was approximately 70%. All traces are from a single platewith A (m=4), B, C, D, E (n=12), and F (n=8).

FIGS. 2A and 2B illustrate the NaCl dose response relationship ofHEK293T cells expressing hENaC α, β, and γ.

FIGS. 3A and 3B illustrate the amiloride dose response relationship ofHEK293T cells expressing hENaC α, β, and γ treated with 50 mM NaCl.

FIG. 4 illustrates the NaCl dose response relationship of HEK293T cellsexpressing ENaC using a voltage imaging plate reader (VIPR). HEK293Tcells were transfected with ENaC subunits expression plasmids (ENaC) ora carrier plasmid (Mock). 24 hours later cells were loaded with amembrane potential dyes and changes in cell fluorescence in response toNa+ stimulation was monitored on VIPRII (Aurora B iosdences). Only cellsexpressing ENaC exhibited a change in response to increases in Na⁺concentration.

FIG. 5 also illustrates the NaCl dose response relationship of HEK2933Tcells expressing human ENaC. HEK293T cells were transfected with ENaCsubunits expression plasmids (ENaC) 24 hours later cells were loadedwith a membrane potential dyes and changes in cell fluorescence inresponse to Na⁺ stimulation was monitored on VIPRII (AuroraBiosciences). Phenamil, an ENaC antagonist inhibited Na⁺-induced changesin fluorescence. Conversely, the Compound “X”, an ENaC enhancer,increased the Na⁺-induced changes in fluorescence and this effect isinhibited by Phenamil.

FIG. 6 shows the effect of increasing concentrations of Phenamil on ENaCactivity. The blue trace: inhibition of ENaC activity by Phenamil. Redtrace: inhibition of ENaC activity by Phenamil in the presence of 100 μMcompound 478354, an ENaC enhancer. The black box contains data showingcompound 478354's effect in the absence of Phenamil. The yellow boxcontains data showing enhanced 478354 effects by the presence ofincreasing concentrations of Phenamil.

FIG. 7A. Distribution of Z′ in the absence of Phenamil. Z¹ is definedas: 1-((3× standard deviation of ENaC responds to 3× standard deviationof ENaC response in the presence of compound 478354)/(mean ENaC activityin the presence of 478354-mean ENaC activity)). Most Z′ values are lessthan 0 indicating that, when used in the high control, 479354 can notprovide a meaningful assay window.

FIG. 7B. Distribution of Z′ in the presence of 0.5 μM Phenamil. Z′ isdefined as: 1-((3× standard deviation of ENaC response+3× standarddeviation of ENaC response in the presence of 478354)/(mean ENaCactivity in the presence of 478354−mean ENaC activity)). Most Z¹ valuesare >0 and ≤1 indicating that as the high control, 478354 can provide ameaningful assay window in the presence of Phenamil.

FIG. 8 illustrates an example of screening oocytes injected with humanENaC cRNA for compounds that increase ENaC activity. For each compoundscreened, a % enhancement factor is calculated. This value correspondsto the magnitude of the current change due to compound divided by themagnitude of the current change due to amiloride multiplied by −100%. Inthis example, two compounds are screened in succession in 7, out of apossible maximum 8, oocytes voltage clamped to −60 mV in the OpusXpresssystem. All 7 oocytes express ENaC, as evidenced by the inhibitoryeffect of amiloride on measured oocyte currents.

FIG. 9 illustrates an example of how the % enhancement factor iscalculated for each oocyte injected with human ENaC cRNA. % enhancementfactors are determined for each compound screened, averaged, andstandard deviations determined. In this case, compound 1 and compound 2correspond to the compounds screened in cells numbered 2 though 8 inFIG. 8.

FIG. 10 illustrates an example of screening oocytes not injected withhuman ENaC cRNA. Compounds have no effect on the activity of ionchannels expressed endogenously in the oocyte membrane, illustratingthat compound activity is ENeC-dependent and attributable to increasedmacroscopic sodium current flowing through ENaC channels. Also thefigure shows that amiloride has no effect on uninjected oocytes due tothe absence of ENaC sodium channel expression.

FIGS. 11A and 11B illustrate examples of I/V curves in the oocytesinjected with human ENaC CRNA or uninjected oocytes in the presence andabsence of compound. In injected eggs, the compound increases the slopeof the I/V curve, whereas in uninjected oocytes the compound has noeffect on the slope of the I/V curve (i.e. the curves in the presenceand absence of compound are identical).

FIG. 12 illustrates an example of an amiloride competition experiment.In oocytes injected with human ENaC cRNA, co-application of amilorideplus compound does not enhance sodium currents flowing through ENaCchannels. This indicates that compounds are working directly on the ENaCchannel; when ENaC channels are closed due to amiloride, compoundscannot enhance ENaC function.

FIG. 13 illustrates an example of dose-response curves for 2 compoundsin oocytes injected with human ENaC cRNA. Compound A is less potent thancompound B as evidenced by its larger EC50 (5.4 uM with compound Acompared to 0.47 uM with compound B) and right-shifted dose-responsecurve.

FIG. 14 schematically illustrates a set of experiments used to examinethe effect of compounds on human ENaC activity in the oocyte expressionsystem using the two-electrode voltage clamp (TEVC) technique.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides assay systems that comprise test cells,preferably recombinant mammalian cells or amphibian oocytes, thatexpress a functional hENaC as well as mammalian cell-based and amphibianoocyte cell-based assays, preferably high or medium throughput, for theprofiling and screening of an epithelial sodium channel (ENaC). Morespecifically, the invention provides human cell lines, e.g., HEK293Tcells, and amphibian oocytes, that express the α, β, and γ subunits ofhENaC that can be used in cell-based assays to screen for ENaCmodulators. Also the invention provides mammalian cells and amphibianoocytes that express a functional ENaC comprised of delta, beta andgamma subunits for use in functionally characterizing ENaC activity, andto identify compounds that either enhance or block salty tasteperception (herein referred to as salty taste modulators). Thesecompounds can be used as ingredients in foods, medicinals and beveragesto enhance, modulate, Inhibit or block salty taste.

However, prior to discussing the invention in more detail the followingdefinitions are provided. It should be otherwise understood that thetechnical terms and phrases have their ordinary meaning, as they wouldbe construed by use of ordinary skill in the art.

Definitions

The term ‘salty taste” or “salty taste perception” as used herein refersto a subject's perception or response to salt taste stimuli. Asdiscussed above, it is believed that hENaC is involved in salty tasteperception, in particular salts that elicit “a salty taste” in humansubjects. Such stimuli include compounds such as NaCl that elicit aresponse in functional ENaCs, preferably hENaC.

The terms “ENaC” subunit protein or a fragment thereof, or a nucleicacid encoding one of three subunits of “ENaC” protein or a fragmentthereof refer to nucleic acids and polypeptides, polymorphic variants,alleles, mutants, and interspecies homologues that: (1) have an aminoacid sequence that has greater than a bout 80% amino acid sequenceidentity, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% or greater amino acid sequence identity, preferably over a region ofover a region of at least about 25, 50, 100, 200, or 500, or more aminoacids, to an amino acid sequence encoded by the nucleic acid sequencecontained in SEQ ID NO:1; 2 or 3; or (2) specifically bind toantibodies, e.g., polyclonal antibodies, raised against an immunogencomprising an amino acid sequence encoded by SEQ ID NO:1, 2, or 7 orimmunogenic fragments thereof, and conservatively modified variantsthereof; or (3) specifically hybridize under stringent hybridizationconditions to an anti-sense strand corresponding to a nucleic acidsequence encoding an ENaC protein, e.g., SEQ ID NO:1, 2, 3 or 7 or theircomplements, and conservatively modified variants thereof, or (4) have anucleic acid sequence that has greater than about 80% sequence identity,85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, orhigher nucleotide sequence identity, preferably over a region of atleast about 25, 50, 100, 200, 500; 1000, or more nucleotides, to SEQ IDNO:1, 2, 3 or 7 or their complements, or (5) is functionally equivalentto the hENaC described herein in a sodium conductance assay whenexpressed in a HEK cell and tested by using two electrode whole cellelectrophysiology or by the change in fluorescence of a membranepotential dye in response to sodium or lithium.

Functionally equivalent ENaC proteins include ENaC subunits with primarysequences different than those identified infra, but which possess anequivalent function as determined by functional assays, e.g., sodiumconductance assays as described infra.

“Determining the functional effect” refers to assaying the effect of acompound that increases- or decreases a parameter that is indirectly ordirectly under the influence of an ENaC polypeptide e.g., functional,physical and chemical effects. Such functional effects include, but arenot limited to, changes in ion flux, membrane potential, currentamplitude, and voltage gating, a as well as other biological effectssuch as changes in gene expression of any marker genes, and the like.The ion flux can include any ion that passes through the channel, e.g.,sodium or lithium and analogs thereof such as radioisotopes. Suchfunctional effects can be measured by any means known to those skilledin the art, e.g., by the use of two electrode electrophysiology orvoltage-sensitive dyes, or by measuring changes in parameters such asspectroscopic characteristics (e.g., fluorescence, absorbance,refractive index), hydrodynamic (e.g., shape), chromatographic, orsolubility properties. Preferably ENaC function will be evaluated byusing two electrode whole cell electrophysiology or by monitoring thechange in fluorescence of a membrane potential dye in response to sodiumor lithium.

“Inhibitors”, “activators”, and “modulators” of ENaC polynucleotide andpolypeptide sequences are used to refer to activating, Inhibitory, ormodulating molecules identified using cell-based assays of ENaCpolynucleotide and polypeptide sequences. Inhibitors are compounds that,e.g., bind to, partially or totally block activity, decrease, prevent,delay activation, inactivate, desensitize, or down regulate the activityor expression of ENaC proteins, e.g., antagonists.

“Activators” are compounds that increase, open, activate, facilitate,enhance activation, sensitize, agonize, or up regulate ENaC proteinactivity. Inhibitors, activators, or modulators also include geneticallymodified versions of ENaC proteins, e.g., versions with alteredactivity, as well as naturally occurring and synthetic ligands,antagonists, agonists, peptides, cyclic peptides, nucleic acids,antibodies, antisense molecules, ribozymes, small organic molecules andthe like. Such assays for inhibitors and activators include, e.g.,expressing ENaC protein in cells, cell extracts, or cell membranes,applying putative modulator compounds, and optionally prior theretocontacting said ENaC protein with a known ENaC inhibitor at aconcentration that results in partial ENaC inhibitors and thendetermining the functional effects of the rotation compound on activity,as described above.

Samples or assays comprising ENaC proteins that are treated with apotential activator, Inhibitor, or modulator are compared to controlsamples without the inhibitor, activator, or modulator to examine theextent of activation, Inhibition or modulation. In one embodiment of theassay, compounds are tested for their effect on the response of cellsprovided with a suboptimal sodium concentration. Control cells, treatedwith the suboptimal concentration of sodium but lacking a compound,typically exhibit a 10-20% of the maximal response. Compounds thatincrease the response of the suboptimal sodium concentration above the10-20% level are putative ENaC enhancers. In contrast, compounds thatreduce the response to below 10% are putative ENaC enhancers.

The term “test compound” or “test candidate” or “modulator” orgrammatical equivalents thereof as used herein describes any molecule,either naturally occurring or synthetic, e.g., protein, oligopeptide(e.g., from about 5 to about 25 amino acids in length, preferably fromabout 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or18 amino acids in length), small organic molecule, polysaccharide, lipid(e.g., a sphingolipid), fatty acid, polynucleotide, oligonucleotide,etc., to be tested for the capacity to modulate ENaC activity. The testcompound can be in the form of a library of test compounds, such as acombinatorial or randomized library that provides a sufficient range ofdiversity. Test compounds are optionally linked to a fusion partner,e.g., targeting compounds, rescue compounds, dimerization compounds,stabilizing compounds, addressable compounds, and other functionalmoieties. Conventionally, new chemical entities with useful propertiesare generated by identifying a test compound (called a “lead compound”)with some desirable property or activity, e.g., enhancing activity,creating variants of the lead compound, and evaluating the property andactivity of those variant compounds. Preferably, high throughputscreening (HTS) methods are employed for such an analysis.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 daltons and less than about 2500 daltons, preferably lessthan about 2000 daltons, preferably between about 100 to about 1000daltons, more preferably between about 200 to about 500 daltons.

“Biological sample” Includes sections of tissues such as biopsy andautopsy samples, and frozen sections taken for histologic purposes. Suchsamples include blood, sputum, tissue, cultured cells, e.g., primarycultures, explants, and transformed cells, stool, urine, etc. Abiological sample is typically obtained from a eukaryotic organism, mostpreferably a mammal such as a primate e.g., chimpanzee or human; cow;dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird;reptile; or fish.

“Compound that inhibits ENaC activity” refers to a compound whichinhibits sodium channel activity, preferably reversibly when thiscompound is contacted with a functional ENaC. Preferred examples of suchcompounds are amiloride and amiloride derivatives such as Phenamil,benzamil, 3¹, 4¹-dichlorobenzamil, ethylisopropylamlorlde;5-(N-4-chlrobenzyl)-2¹, 4¹ dimethyl-benzamil,5-(N-methyl-N-guanidinocarbonylmethyl) amiloride; 5-(N,N-hexa-myethylene) amiloride; 5-(N-ethyl-N-isopropyl) amiloride (EIPA);5-(N-4-chloro-benzyl) 2¹, 4¹ dimethylbenzamil, 2¹, 4′-dimethylbenzamil;2¹, 3¹-benzo-benzamil; and the like.

“Amiloride derivative” refers to a compound having a structure similarto amiloride which inhibits ENaC function. Typically such derivativesare substituted on the guanidine substituent (e.g., Phenamil) or on the5-N position (e.g., ethylisopropylamiloride).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 80% identity, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or higher identity over a specified region (e.g.,nucleotide sequences SEQ ID NO: 1, 2, 3 or 7), when compared and alignedfor maximum correspondence over a comparison window or designatedregion) as measured using a BLAST or BLAST 2.0 sequence comparisonalgorithms with default parameters described below, or by manualalignment and visual inspection (see, e.g., NCBI web site(www.ncbi.nlm.nih.gov) or the like). Such sequences are then said to be“substantially identical.” This definition also refers to, or may beapplied to, the compliment of a test sequence. The definition alsoincludes sequences that have deletions and/or additions, as well asthose that have substitutions. As described below, the preferredalgorithms can account for gaps and the like. Preferably, identityexists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, Includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example; of an algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino add. Amino add mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but those functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleic addswhich encode identical or essentially identical amino acid sequences, orwhere the nucleic acid does not encode an amino acid sequence, toessentially identical sequences. Because of the degeneracy of thegenetic code, a large number of functionally identical nucleic acidsencode any given protein. For instance, the codons GCA, GCC, GCG and GCUall encode the amino acid alanine. Thus, at every position where analanine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein that encodes a polypeptide also describes every possiblesilent variation of the nucleic acid. One of skill will recognize thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for m ethionine, and TGG which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Accordingly, each silent variation of a nucleic acid that encodes apolypeptide is implicit in each described sequence with respect to theexpression product, but not with respect to actual probe sequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologous, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan. (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see. e.g., Creighton. Proteins (1984)). As notedpreviously, the invention embraces cells that express ENaC subunitpolypeptides having primary sequences different than those disclosed inthe subject application that are functionally equivalent in appropriateassays, e.g., using whole cell sodium conductance assays described indetail infra.

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered three-dimensional structures within a polypeptide. Thesestructures are commonly known as domains, e.g., transmembrane domainspore domains; and cytoplasmic tail domains. Domains are portions of apolypeptide that form a compact unit of the polypeptide and aretypically to 350 amino acids long. Exemplary domains includeextracellular domains, transmembrane domains, and cytoplasmic domains.Typical domains are made up of sections of lesser organization such asstretches of α-sheet and β-helices. “Tertiary structure” refers to thecomplete three-dimensional structure of a polypeptide monomer.“Quaternary structure” refers to the three dimensional structure formedby the noncovalent association of independent tertiary units.Anisotropic terms are also known as energy terms.

A particular nucleic acid sequence also implicitly encompasses “splicevariants.” Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant of thatnucleic acid. “Splice variants,” as the name suggests, are products ofalternative splicing of a gene. After transcription, an initial nucleicacid transcript may be spliced such that different (alternate) nucleicacid splice products encode different polypeptides. Mechanisms for theproduction of splice variants vary, but include alternate splicing ofexons. Alternatively polypeptides derived from the same nucleic acid byread-through transcription are also encompassed by this definition. Anyproducts of a splicing reaction, including recombinant forms of thesplice products, are included in this definition.

ENaC nucleic acid sequences also include single nucleotide polymorphismswhich encode ENaC subunits that are functionally equivalent to the ENaCpolypeptides disclosed herein when assayed using appropriate assays, inthe sodium conductance assays described herein.

Membrane potential dyes or voltage-sensitive dyes refer to a molecule orcombinations of molecules that change fluorescent properties uponmembrane depolarization. These dyes can be used to detect the changes inactivity of an ion channel such as ENaC expressed in a cell.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example; useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins whichcan be made detectable, e.g., by incorporating a radiolabel into thepeptide or used to detect antibodies specifically reactive with thepeptide.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, Indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all. In the present invention thistypically refers to cells that have been transfected with nucleic acidsequences that encode one or more ENaC subunits.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein). The term “heterologous” when used with reference tocellular expression of a gene, cDNA, mRNA or protein indicates that thegene, cDNA, mRNA, or protein is not normally expressed in the cell or isfrom another species than the original source of the cells.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SOS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C. with wash in 0.2×SSC, and 0.1% SDSat 65° C. The wash and hybridization steps are generally carried out for%, 1, 2, 5, 13, 15, 30, 60 or more minutes, and more typically for about30 seconds to 2 minutes.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al.

For PCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to a bout65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al. (1990) PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y.).

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes. IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding.

Particularly, such an antibody includes one which specifically binds toan ENaC disclosed herein, or a mixture of antibodies that specificallybind such ENaC polypeptides.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, often in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein atleast two times the background and more typically more than 10 to 100times background. Specific binding to an antibody under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to ENaCsubunit proteins, e.g., the ENaC alpha, beta, gamma or delta subunits asencoded by SEQ ID NO:1, 2, 3, or 7, polymorphic variants, alleles,orthologs, and conservatively modified variants, or splice variants, orportions thereof, can be s elected to obtain only those polyclonalantibodies that are specifically immunoreactive with ENaC subunitproteins i.e., ENaC alpha, beta, gamma or delta subunits, e.g., thosehaving the amino acid sequences contained in SEQ ID NO.: 4, 5, 6 or 8,and not with other proteins. This selection may be achieved bysubtracting out antibodies that cross-react with other molecules. Avariety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select antibodiesspecifically immunoreactive with a protein (see, e.g., Harlow & Lane,Antibodies, A Laboratory Manual (1988) for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity).

Assays for Proteins that Modulate ENaC Activity

High or medium throughput functional genomics assays can be used toidentify modulators of ENaC which block, inhibit, modulate or enhancesalty taste. Such assays can, e.g., monitor changes in cell surfacemarker expression, changes in intracellular ions, or changes in membranecurrents using either cell lines or primary cells or oocytes. Typically,the cells are contacted with a cDNA or a random peptide library (encodedby nucleic acids). The cDNA library can comprise sense, antisense, fulllength, and truncated cDNAs. The peptide library is encoded by nucleicacids. The effect of the cDNA or peptide library on the phenotype of thecells is then monitored, using an assay as described above. The effectof the cDNA or peptide can be validated and distinguished from somaticmutations, using, e.g., regulatable expression of the nucleic acid suchas expression from a tetracycline promoter. cDNAs and nucleic acidsencoding peptides can be rescued using techniques known to those ofskill in the art, e.g., using a sequence tag.

Proteins interacting with the peptide or with the protein encoded by thecDNA (e.g., SEQ ID NO: 1, 2, or 7) can be isolated using a yeasttwo-hybrid system, mammalian two hybrid system, or phage display screen,etc. Targets so identified can be further used as bait in these assaysto identify additional components that may interact with the ENaCchannel which members are also targets for drug development (see, e.g.,Fields et al., Nature 340:245 (1989); Vasavada et al., Proc. Nat'l Acad.Sc. USA 88:10686 (1991); Fearon et al., Proc. Nat'l Acad. Sci. USA89:7958 (1992); Dang et al., Mol. Cell. Biol. 11:954 (1991); Chien etal., Proc. Nat'l Acad. Sc. USA. 9578 (1991); and U.S. Pat. Nos.5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463).

Suitable cells and cell lines that express ENaC proteins include, by wayof example, kidney epithelial cells, lung epithelial cells, tasteepithelial cells and other mammalian epithelial cells, preferably human,and oocytes, preferably amphibian oocytes, most preferably Xenopusoocytes.

Isolation of Nucleic Acids Encoding ENaC Proteins

This invention relies, in part, on routine techniques in the field ofrecombinant genetics. Basic texts disclosing the general methods of usein this invention Include Sambrook and Russell, Molecular Cloning, ALaboratory Manual (3rd ed. 2001); Kregler, Gene Transfer and Expression:A Laboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

Nucleic adds that encode ENaC subunits, polymorphic variants, orthologs,and alleles that are substantially identical to an amino acid sequenceencoded by SEQ ID NQ: 1, 2, 3 or 7 as well as other ENaC family members,can be isolated using ENaC nucleic acid probes and oligonucleotidesunder stringent hybridization conditions, by screening libraries.Alternatively, expression libraries can be used to done ENaC subunitprotein, polymorphic variants, orthologs, and alleles by detectingexpressed homologous immunologically with antisera or purifiedantibodies made against human ENaC or portions thereof.

To make 1 cDNA library, one should choose a source that is rich in ENaCRNA. The mRNA is then made into cDNA using reverse transcriptase,ligated into a recombinant vector, and transfected into a recombinanthost for propagation, screening and cloning. Methods for making andscreening cDNA libraries are well known (see, e.g., Gubler & Hoffman,Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

Alternatively, ENaC cRNA encoding human ENaC subunits may be generatedfrom α, β, γ or Δ human ENaC DNA plasmids using T7 RNA polymers totranscribe cRNA in vitro from DNA linearized with appropriaterestriction enzymes and the resultant cRNA microinjected into suitablecells, e.g., oocytes, preferably frog oocytes.

An alternative method of isolating ENaC subunit nucleic acid and itsorthologs, alleles, mutants, polymorphic variants, and conservativelymodified variants combines the use of synthetic oligonucleotide primersand amplification of an RNA or DNA template (see U.S. Pat. Nos.4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications (Innis et al., eds, 1990)). Methods such as polymerasechain reaction (PCR) and ligase chain reaction (LCR) can be used toamplify nucleic acid sequences of human ENaC directly from mRNA, fromcDNA, from genomic libraries or cDNA libraries. Degenerateoligonucleotides can be designed to amplify ENaC homologs using thesequences provided herein. Restriction endonuclease sites can beincorporated. Into the primers. Polymerase chain reaction or other invitro amplification methods may also be useful, for example, to donenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of ENaCencoding mRNA in physiological samples, for nucleic acid sequencing, orfor other purposes. Genes amplified by the PCR reaction can be purifiedfrom agarose gels and cloned into an appropriate vector.

Gene expression of ENaC subunits can also be analyzed by techniquesknown in the art, e.g., reverse transcription and amplification of mRNA,isolation of total RNA or poly A⁺ RNA, northern blotting, dot blotting,in situ hybridization, RNase protection, high density polynucleotidearray technology, e.g., and the like.

Nucleic acids encoding ENaC subunit proteins can be used withhigh-density oligonucleotide array technology (e.g., GeneChip™) toidentify ENaC protein, orthologs, alleles, conservatively modifiedvariants, and polymorphic variants in this invention. In the case wherethe homologs being identified are linked to modulation of T cellactivation and migration, they can be used with GeneChip™ as adiagnostic tool in detecting the disease in a biological sample, see,e.g., Gunthand et al., AIDS Res. Hum. Retroviruses 14: 869-876 (1998);Kozal et al., Nat. Med. 2:753-759 (1996); Matson et al., Anal. Biochem.224:110-106 (1995); Lockhart et al., Nat. Biotechnol. 14:1675-1680(1996); Gingeras et al., Genome Res. 8:435-448 (1998); and Hacia at al.,Nucleic Acids Res. 26:3865-3866 (1998).

The genes encoding ENaC subunits preferably human ENaC subunits aretypically cloned into intermediate vectors before transformation intoprokaryotic or eukaryotic cells for replication and/or expression. Theseintermediate vectors are typically prokaryotic vectors, e.g., plasmids,or shuttle vectors.

1. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding hENaC subunit, one typically subclones the hENaC subunitnucleic acid sequence into an expression vector that contains a strongpromoter to direct transcription, a transcription/translationterminator, and if for a nucleic acid encoding a protein, a ribosomebinding site for translational initiation. Suitable bacterial promotersare well known in the art and described, e.g., in Sambrook et al., andAusubel et al, supra. Bacterial expression systems for expressing theENaC subunit protein are available in, e.g., E. coli, Bacillus sp., andSalmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature302:643-545 (1983). Kits for such expression systems are commerciallyavailable. Eukaryotic expression systems for mammalian cells, yeast,Xenopus oocytes and insect cells are well known in the art and are alsocommercially available.

In a preferred embodiment of the invention, an oocyte expression systemis used to express a functional human ENaC and to examine the effects ofspecific compounds on sodium transport through ENaC channels. TheXenopus oocyte expression system has previously been used for theexpression of ion channels, including ENaC, and in functional studies(Dascal, CRC Crit. Rev. Biotech. (1987) 22(4):317-387; Wagner, et al.,Cellular Physiology and Biochemistry (2000) 10:1-12; and Canessa et al.,Nature (1994) 367:463-467). In still another embodiment retroviralexpression systems may be used in the invention. In another embodimenttransient expression systems may be utilized with plasmid-based vectorsthat are commercially available such as pcDNA 3 and derivatives thereof

Selection of the promoter used to direct expression of a heterologousnucleic acid depends on the particular application. The promoter ispreferably positioned about the same distance from the heterologoustranscription start site, as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the ENaC subunitencoding nucleic acid in host c ells. A typical expression cassette thuscontains at least one promoter operably linked to a nucleic acidsequence encoding a ENaC subunit(s) and signals required for efficientpolyadenylation of the transcript, ribosome binding sites, andtranslation termination. Additional elements of the cassette may includeenhancers and, if genomic DNA is used as the structural gene, intronswith functional splice donor and acceptor site.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as MBP, GST, and LacZ. Epitope tags can also beadded to recombinant proteins to provide convenient methods ofisolation, e.g., c-myc. Sequence tags may be included in an expressioncassette for nucleic acid rescue. Markers such as fluorescent proteins,green or red fluorescent protein, β-gal, CAT, and the like can beincluded in the vectors as markers for vector transduction.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, retroviral vectors, and vectorsderived from Epstein-Barr virus. Other exemplary eukaryotic vectorsinclude pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, andany other vector allowing expression of proteins under the direction ofthe CMV promoter, SV40 early promoter, SV40 late promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

Expression of proteins from eukaryotic vectors can be also regulatedusing inducible promoters. With inducible promoters, expression levelsare tied to the concentration of inducing agents, such as tetracyclineor ecdysone, by the incorporation of response elements for these agentsinto the promoter. Generally, high level expression is obtained frominducible promoters only in the presence of the inducing agent; basalexpression levels are minimal.

In one embodiment, the vectors of the invention may have a regulatablepromoter, e.g., tet-regulated systems and the RU-486 system (see, e.g.,Gossen & Bujard, PNAS 89:5547 (1992); Oligino et al., Gene Ther.5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997); Neering etal., Blood 88:1147-1155 (1996); and Rendahl et al., Nat. Biotechnol.16:757-761 (1998)). These impart small molecule control on theexpression of the candidate target nucleic acids. This beneficialfeature can be used to determine that a desired phenotype is caused by atransfected cDNA rather than a somatic mutation.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase and dihydrofolate reductase. Alternatively,high yield expression systems not involving gene amplification are alsosuitable, such as using a baculovirus vector in insect c ells, with aENaC encoding sequence under the direction of the polyhedrin promoter orother strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods may be used to produce bacterial,mammalian, yeast or insect cell lines that express large quantities ofENaC protein, which are then purified using standard techniques (see,e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide toProtein Purification, in Methods in Enzymology, vol. 182 (Deutscher,ed., 1990)). Transformation of eukaryotic and prokaryotic cells areperformed according to standard techniques (see, e.g., Morrison, J.Bact. 132:349-351 (1977); Clark-Curtis & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983). Alternatively, in a preferredembodiment of the invention, oocytes that express human ENaC subunitsare produced by microinjection of cRNA encoding said subunits therein.

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,biolistics, liposomes, lipids optimized for DNA transfection,microinjection, plasma vectors, viral vectors and any of the other wellknown methods for introducing cloned genomic DNA, cDNA, synthetic DNA orother foreign genetic material into a host cell (see, e.g., Sambrook etal., supra). It is only necessary that the particular geneticengineering procedure used be capable of successfully introducing atleast one ENaC subunit gene into a host cell, preferably mammaliancapable of expressing functional ENaC.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofENaC subunit(s). In one embodiment, the cells are transientlytransfected with all three hENaC genes using lipid-based transfectionand cultured for 24-48 hours prior to performing the screen for ENaCmodulators.

As noted previously, a preferred embodiment of the invention comprisesan oocyte expression system. Those methods generally include frogsurgery, oocyte isolation, cRNA preparation and oocytes microinjection.General procedures for frog surgery and ocyte isolation areconventionally known in the art. (See, Marcus-Sekur, et al., Methods inEnzymol. 152:284-288 (1987); Goldin, Methods in Enzymol. 207:266-279.)Likewise, methods for preparing cRNA are also well known and arereported, e.g., in Swansen, et al., Meth. Enzymol. 207:310-319 (1912),Golden, et al., Meth. Enzymol. S217:279-297 (1992). The resultant cRNAis then microinjected into frog oocytes by standard methods. (See,Molten, et al., Moth. Enzymol. 254:458-466 (1975); Hitchcock et al.,Meth. Enzymol. 152:276-284 (1987).

Assays for Modulators of ENaC Protein

A. Assays

Modulation of an ENaC protein can be assessed using a variety of assays;preferably cell-based models as described above. Such assays can be usedto test for inhibitors and activators of ENaC, which modulate, block,enhance or inhibit salty taste perception.

Preferably, the ENaC will be comprised of three subunits, alpha (ordelta), beta and gamma and preferably the human ENaC subunit encoded bythe encoded by SEQ ID NO: 1, 2, 3 or 7 or a human ortholog aconservatively modified variant thereof. Alternatively, the ENaC of theassay will be derived from a non-human epithelial cell. Generally, theamino acid sequence identity of each respective subunit will be at least80%, preferably at least 85%, or 90%, most preferably at least 95%,e.g., 96%, 97%, 98% or 99% to the polypeptide encoded by SEQ ID NO: 1,2, 3 or 7.

Measurement of the effect of a candidate comprised or an ENaC protein orcell expressing ENaC protein, either recombinant or naturally occurring,can be performed using a variety of assays, as described herein.Preferably to identify molecules capable of modulating ENaC, assays areperformed to detect the effect of various candidate modulators on ENaCactivity in an amphibian oocyte or mammalian cell that expresses afunctional ENaC. Preferably, such assays will initially contact ENaCwith a known ENaC inhibitor prior to the addition of at least oneputative ENaC modulator, e.g., ENaC enhancer. Preferably, the inhibitorwill be amiloride or an amiloride derivative such as Phenamil.

The channel activity of ENaC proteins can be assayed using a variety ofassays to measure changes in ion fluxes including patch clamptechniques, measurement of whole cell currents, radiolabeled ion fluxassays, and fluorescence assays using voltage-sensitive dyes (see. e.g.,Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Daniel etal., J. Pharmacol. Meth. 25:185-193 (1991); Hoevinsky et al., J.Membrane Biol. 137:59-70 (1994)) and ion-sensitive dyes. For example,nucleic acids encoding one or more subunits of an ENaC protein orhomologue thereof can be injected into Xenopus oocytes. Channel activitycan then be assessed by measuring changes in membrane current. One meansto obtain electrophysiological measurements is by measuring currentsusing patch clamp techniques. e.g., the “cell-attached” mode, the“inside-out” mode, and the “whole cell” mode (see, e.g., Ackerman etal., New Engl. J. Med. 336:1575-1595, 1997). Whole cell currents can bedetermined using standard methodology such as that described by Hamil etal., Pflugers. Archiv. 391:185 (1981).

Channel activity is also conveniently assessed by measuring changes inintracellular ion levels for example using ion sensitive dyes.

The activity of ENaC polypeptides can be also assessed using a varietyof other assays to determine functional, chemical, and physical effects,e.g., measuring the binding of ENaC polypeptides to other molecules,including peptides, small organic molecules, and lipids; measuring ENaCprotein and/or RNA levels, or measuring other aspects of ENaCpolypeptides, e.g., transcription levels, or physiological changes thataffects ENaC activity. When the functional consequences are determinedusing intact cells or animals, one can also measure a variety of effectssuch as changes in cell growth or pH changes or changes in intracellularsecond messengers such as IP3, cGMP, or cAMP, or components orregulators of the phospholipase C signaling pathway. Such assays can beused to test for both activators and inhibitors. Modulators thusidentified are useful for, e.g., as flavorants in foods, beverages andmedicines.

Cell-Based Assays

In another embodiment, at least one ENaC subunit protein is expressed ina cell, and functional, e.g., physical and chemical or phenotypic,changes are assayed to identify ENaC modulators. Cells expressing ENaCproteins can also be used in binding assays. Any suitable functionaleffect can be measured, as described herein. For example, changes inmembrane potential, changes in intracellular ion levels, and ligandbinding are all suitable assays to identify potential modulators using acell based system. Suitable cells for such cell-based assays includeboth primary cells, e.g., taste epithelial cells that expresses an ENaCprotein and cultured cell lines such as HEK293T cells that express anENaC. Another preferred expression system will comprise amphibianoocytes. As noted, these assays will preferably initially contact theENaC expression cell line, e.g., amphibian oocytes or HEK293 with aknown ENaC inhibitor, e.g., amiloride or an amiloride derivative such asPhenamil at a concentration that partially inhibits ENaC function, priorto contacting the cell line with at least one putative ENaC modulator.The ENaC protein can be naturally occurring or recombinant. Also, asdescribed above, fragments of ENaC proteins or chimeras with ion channelactivity can be used in cell based assays.

In yet another embodiment, cellular ENaC polypeptide levels aredetermined by measuring the level of protein or mRNA. The level of ENaCprotein or proteins related to ENaC ion channel activation are measuredusing immunoassays such as western blotting, ELISA and the like with anantibody that selectively binds to the ENaC polypeptide or a fragmentthereof. For measurement of mRNA, amplification, e.g., using PCR, LCR,or hybridization assays, e.g., Northern hybridization, RNase protection,dot blotting, is preferred. The level of protein or mRNA is detectedusing directly or indirectly labeled detection agents, e.g.,fluorescently or radioactively labeled nucleic acids, radioactively orenzymatically labeled antibodies, and the like, as described herein.

Alternatively, ENaC expression can be measured using a reporter genesystem. Such a system can be devised using an ENaC protein promoteroperably linked to a reporter gene such as chloramphenicolacetyltransferase, firefly luciferase, bacterial luciferase,fgalactosidase and alkaline phosphatase. Furthermore, the protein ofinterest can be used as an indirect reporter via attachment to a secondreporter such as red or green fluorescent protein (see, e.g., Mistill &Spector, Nature Biotechnology 15:961-964 (1997)). The reporter constructis typically transfected into a cell. After treatment with a potentialmodulator, and preferably prior thereto treatment with a known ENaCinhibitor, e.g., Phenamil, the amount of reporter gene transcription,translation, or activity is measured according to standard techniquesknown to those of skill in the art.

In another embodiment, a functional effect related to signaltransduction can be measured. An activated or inhibited ENaC will-alterthe properties of target enzymes, second messengers, channels, and othereffector proteins. Assays for ENaC activity include cells that areloaded with ion or voltage sensitive dyes to report channel activity,e.g., by observing membrane depolarization or sodium influx. Assays fordetermining activity of such receptors can also use known antagonistsfor ENaC, such as amiloride or phenamil; as controls to assess activityof tested compounds. In assays for identifying modulatory compounds(e.g., agonists, antagonists), changes in the level of ions in thecytoplasm or membrane potential will be monitored using an ion sensitiveor membrane potential fluorescent indicator, respectively. Among theion-sensitive indicators and voltage probes that may be employed arethose disclosed in the Molecular Probes 2002 Catalog: (www.probes.com).and specific compounds disclosed infra.

A preferred assay system will use frog oocytes injected with ENaC cRNAswhich are contacted with a test compound and then analyzed by thetwo-electrode voltage clamp electrophysiological recording technique.(See Stuhmer, Meth. Enzymol. 207:319-339 (1992); Wagner et al., CellularPhysiology and Biochemistry 10:1-12 (2000)).

Electrophysiologicall Assay

As noted, a preferred assay for identification of compounds thatmodulate, i.e., enhance, inhibit or block ENaC comprises anelectrophysiological assay that monitors changes in electrical currentin cells that express human ENaC subunits that are contacted with atleast one putative ENaC modulator (enhancer or inhibitor). These assaysmay use any cell that expresses a functional ENaC. In the preferredembodiment, the cells will comprise oocytes, preferably frog oocytes,mammalian cells, yeast cells or insect cells, or another expressionsystem that is suitable for expressing a functional ENaC ion channel.Preferably, the expression system will exhibit robust and rapid humanENaC sodium channel expression and desirably will not express any orvery few endogenous ion channels, thereby facilitating theidentification of compounds that specifically modulate ENaC sodiumchannel function. Thereby, an undesirable background response isminimized or eliminated. Moreover, robust cells, such as oocytes, aredesirable as this enables the cells to be reused in assays according tothe invention. Oocytes have been reported previously to rapidly androbustly express other functional ion channels including ENaCs (Pascal.CRC Crit. Rev. Biotech. 22(4):317-87 (1987); Wagner et al., CellPhysiol. Biochem. 10:1-12 (2000); Canessa et al., Nature 367:463-467(1994)).

A particularly preferred electrophysiologlcal assay is a moderatethroughput assay that measures ENaC sodium channel function in frogoocytes by the two-electrode voltage clamp technique. This robust, fastexpression system provides for the expression of ˜1 million ENaCchannels in an oocyte membrane after only about 18-24 hours. Moreover,because oocytes are relatively large (1 mm in diameter, relatively largecompared to most mammalian cells), they are easy to handle and workwith.

Based on these advantages, a single oocyte can be used to obtainmultiple and repetitive electrophysiological recording. Also, an oocytetypically expresses few endogenous channels, and expression is at levelsbelow that which cause high background relative to the background seenin some other expression systems, e.g., HEK293T cells. Thereby, oocytesallow for repeated direct measurement of the effect of target compoundson ENaC sodium channel function.

In a preferred two-electrode voltage clamp assay according to theinvention (exemplified in detail in the Example 4 infra), frog oocytesthat have been microinjected with ENaC α, β, and γ human ENaC cRNAs (orδ, β and γ) human ENaC cRNAs) are transferred to glass scintillationvials and incubated under appropriate conditions to facilitate ENaCprotein expression.

After ENaC sodium ion channel expression is obtained, typically around24 hours post-cRNA microinjection, ENaC function is measured accordingto the two-electrode voltage clamp technique using an appropriatetwo-electrode voltage measuring device, e.g., OpusXpress 6000A paralleloocyte voltage clamp system (Axon Instruments). The two-electrodevoltage clamp technique measures the macroscopic electrical currentflowing across the entire oocyte membrane through the ENaC sodium ionchannels. Oocytes are punctured with a voltage-sensing electrode and acurrent sensing electrode; the voltage, or potential difference acrossthe oocyte membrane, is clamped to a particular value using thevoltage-sensing electrode and the current, or the flow of ions acrossthe oocyte membrane, required to maintain the voltage is measured usingthe current-sensing electrode. The OpusXpresss system is one example ofa commerically available two-electrode voltage measuring device which issemi-automated and which comprises a workstation that permitselectrophysiological recordings to be made from 8 oocytessimultaneously. This system also provides for automated oocyteimpalement and delivery of target compounds by a computer-controlledfluid handler that delivers compound into 96-well compound plates. Thissystem can best be described as a medium or moderate-throughput systemas it allows for the evaluation of about 60 compounds per week. Ofcourse more compounds can be screened by the addition of other voltagemeasuring devices, as described.

In this assay system, ENaC enhancers will result in an increase incurrent passing through the ENaC channels in the oocyte membrane. Thisvalue is calculated by a standard formula provided infra in (Example 4).Such assays also may include appropriate negative controls, e.g.,amiloride, which is a known ENaC inhibitor that blocks sodium transportthrough ENaC channels. Therefore, this compound functions both as aninternal control to verify that oocytes express functional ENaC, and, inoocytes exhibiting amiloride inhibition, allows for the screening ofputative ENaC enhancers after amiloride compound is applied (if thetarget compound is an ENaC enhancer it will result in an increase incurrent passing through ENaC channels in the oocyte membrane).

Desirably, a % enhancement factor is calculated for each enhancer. Forexample, a 100% enhancer increases ENaC activity 100% relative to thebasal control value (no compound).

Negative controls are also desirably performed to confirm that oocyteswhich are not injected with ENaC cRNAs do not exhibit the same effects.

As discussed in greater detail in Example 4 infra, more complex analysesare also desirably performed on compounds that exhibit maximal %enhancement valves e.g., current/voltage (I/V) curves, amiloridecompetitive experiments and dose-response curves to determine theconcentration at which the compound exhibits half-maximal activity (EC50value). These experiments will further confirm that the effect of thecompound is ENaC-specific.

These assays will provide for the identification of ENaC modulators,preferably ENaC enhancers, which may be used as additives for foods,beverages, pharmaceuticals and the like in order to modulate the saltytaste associated therewith. Desirably, an ENaC enhancer will exhibit atleast 20% enhancement factor, more preferably at least 50% and even morepreferably at least an 100% enhancement factor. These oocyte-basedassays are discussed in further detail as well as the intrinsicadvantages associated therewith in Example 4 of this application.

Animal Models

Animal models that express hENaC also find use in screening formodulators of salty taste. Similarly, transgenic animal technologyincluding gene knockout technology, for example as a result ofhomologous recombination with an appropriate gene targeting vector, orgene overexpression, will result in the absence or increased expressionof the ENaC protein. The same technology can also be applied to makeknockout cells. When desired, tissue-specific expression or knockout ofthe ENaC protein may be necessary. Transgenic animals generated by suchmethods find use as animal models of responses to salty taste stimuli.

Knockout cells and transgenic mice can be made by insertion of a markergene or other heterologous gene into an endogenous ENaC gene site in themouse genome via homologous recombination. Such mice can also be made bysubstituting an endogenous ENaC with a mutated version of the ENaC gene,or by mutating an endogenous gene.

A DNA construct is introduced into the nuclei of embryonic stem cells.Cells containing the newly engineered genetic lesion are injected into ahost mouse embryo, which is re-implanted into a recipient female. Someof these a embryos develop into chimeric mice that possess germ cellspartially derived from the mutant cell line. Therefore, by breeding thechimeric mice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capeochi et al., Science 244:1288(1989)). Chimeric targeted mice can be derived according to Hogan etal., Manipulating the Mouse Embryo: A Laboratory Manual, Cold SpringHarbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells.A Practical Approach, Robertson, ed., IRL Press, Washington, D.C.,(1987).

B. Modulators

The compounds tested as modulators of ENaC protein can be any smallorganic molecule, or a biological entity, such as a protein, e.g., anantibody or peptide, a sugar, a nucleic acid, e.g., an antisenseoligonucleotide or a ribozyme, or a lipid. Alternatively, modulators canbe genetically altered versions of an ENaC protein. Typically, testcompounds will be small organic molecules, peptides, lipids, and lipidanalogs. Preferably, the tested compounds are safe for humanconsumption.

Essentially any chemical compound can be used as a potential modulatoror ligand in the assays of the invention, although most often compoundscan be dissolved in aqueous or organic (especially DMSO-based) solutionsare used. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including ChemDiv (San Diego, Calif.), Sigma-Aldrich (St. Louis, Mo.),Fluka Chemika-Biochemica-Analytika (Buchs Switzerland) and the like.

In the preferred embodiment, moderate or high throughput screeningmethods involve providing a small organic molecule or peptide librarycontaining a significant number of potential ENaC modulators (potentialactivator or inhibitor compounds). Such “chemical libraries” are thenscreened in one or more assays, as described herein, to identify thoselibrary members (particular chemical species or subclasses) that displaya desired characteristic activity. The compounds thus identified canserve as conventional “lead compounds” or can themselves be used aspotential or actual products. As noted, the preferred oocyte two-voltageclamp electrode system (a single device) permits about 60 compounds tobe tested per week.

A combinatorial chemical library is a collection of diverse chemical,compounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Scd. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathlazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Blosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals. Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

Foods and Beverage Compositions Containing ENaC Modulatory CompoundIdentified Using Disclosed Assays

The compounds identified using the disclosed assays, e.g., theelectrophysiological (two electrode voltage-clamp technique) assays andfluorescence cell-based assay disclosed in the examples, are potentiallyuseful as ingredients or flavorants in ingestible compositions, i.e.,foods and beverages as wells as orally administered medicinals.Compounds that modulate or enhance salty taste perception can be usedalone or In combination as flavorants in foods or beverages. In thepreferred application, the modulator will be incorporated into a food orbeverage with a reduced level of sodium and the salty taste of theresulting product will be similar to that of the high sodium product.Examples of such foods and beverages include snack foods such aspretzels, potato chips, crackers, soups, dips, soft drinks, packagedmeat products, among others.

Alternatively, compounds that block or inhibit salty taste perceptioncan be used as ingredients or flavorants in foods that naturally containhigh salt concentrations in order to block or camouflage the salty tastethereof.

The amount of such compound(s) will be an amount that yields the desireddegree of salty taste perception. Of course compounds used in suchapplications will be determined to be safe for human consumption and tobe acceptable in human taste tests.

Preferred Assay Embodiment Using Phenamil or Equivalent

As disclosed supra, one of the preferred embodiments of the inventionwill comprise contacting a test cell expressing a functional ENaC withat least one putative modulator compound in the presence of a membranepotential dye, and preferably prior thereto contacting said test cellwith at least one compound known to modulate (inhibit) ENaC function,preferably an amiloride derivative such as Phenamil and monitoring theactivity of the ENaC expressed by the test cell to determine the extentof ENaC modulation. As noted, the addition of an ENaC inhibitor prior tothe test compound improves assay results. This inhibitor. e.g.,Phenamil. Is used at a concentration that at least partially inhibitsENaC function. The method can further comprise evaluating the putativemodulator compound for in vivo effects on salty taste perception (e.g.,performing tasting experiments to determine the in vivo effect on saltytaste perception). For example, cDNAs encoding the ENaC subunits arecloned from human kidney cell cDNA, human lung cell cDNA, or human tastecell cDNA. As mentioned above, native ENaC is a multimeric proteinconsisting of three subunits (alpha or delta, beta, and gamma). ENaCfunctions as a constitutively active Na⁺ selective cation channel, isfound in taste buds as well as other tissues, and is a candidate humansalt receptor underlying the physiological perception of salty taste.

In another preferred embodiment of the invention, such a method iscarried out in a high throughput assay format using multi-well platesand a fluorescence intensity plate reader (e.g., Aurora Biosciences VIPRInstrument or Molecular Device's FLIPR instrument). The test cells maybe seeded, dye-loaded, optionally, preferably initially contacting thetest cells with a known ENaC inhibitor at a concentration whereby ENaCfunction is at least partially and preferably reversibly inhibited,thereafter contacting said test cell with at least one test compound,and monitoring fluorescence intensity in the same multi-well plate. Suchan assay format can reliably detect both activation or inhibition ofENaC function, providing a robust screen for compounds that could eitherenhance or block channel activity. The assay described above has beenoptimized to identify ENaC enhancers. The assay described herein thushas advantages over existing assays, such as those described above, inthat a human ENaC is utilized, mammalian cells are employed and theassay can be run in standard multi-well (e.g., 96, 384, or 1536 well)plates in high-throughput mode. (However, as discussed above, mammaliancells possess some disadvantageous properties, e.g., they may expressendogenous ion channels at levels resulting in undesirable backgroundlevels.)

In this preferred embodiment of the invention, cells, preferablymammalian cells, will be produced that functionally express at least thealpha (or delta) subunit of ENaC. In preferred embodiments, all threesubunits of hENaC (α or δ, β, and γ) are expressed either transiently orstably. The ENaC subunit(s) employed can be naturally occurring forms,variants containing SNPs, alternatively spliced forms, combinations offorms or any functional variants known in the art (see e.g., accessionnumbers P37088, P51168, P51170, and P51172). Preferably, the ENaC willbe comprised of the human alpha, beta and gamma ENaC subunits encoded bythe nucleic acid sequence in SEQ ID NO. 1, 2, 3 or the human beta, gammaand delta ENaC subunits encoded by SEQ ID NO. 2, 3 and 7. The mammaliancells can be any type known in the art such as COS, CHO, BHK, MDCK,HEK293, or HEK293T (human embryonic kidney cells expressing the largeT-cell antigen). Preferably, the cell is HEK293T. The cells can betransfected using standard methods known in the art, such as but notlimited to Ca²⁺ phosphate or lipid-based systems, or methods previouslymentioned.

These transfected cells are then preferably seeded into multi-wellculture plates. Functional expression is then allowed to proceed for atime sufficient to reach at least about 70% confluence, more preferablyto at least about 80% confluence or to form a cell layer dense enough towithstand possible fluid perturbations caused by compound addition.Generally, an incubation time of at least 24 hours will be sufficient,but can be longer as well. The cells are then washed to remove growthmedia and incubated with a membrane-potential dye for a time sufficientto allow the dye to equilibrate across the plasma membranes of theseeded cells. One of skill in the art will recognize that the dyeloading conditions are dependent on factors such as cell type, dye type,incubation parameters, etc. In one embodiment, the dye may be used atabout 2 μM to about 5 μM of the final concentration. Further, theoptimal dye loading time may range from about 30 to about 80 minutes at37° C. for most cells. In the preferred embodiment, the membranepotential dyes are from Molecular Devices (cat# R8034). In otherembodiments, suitable dyes include e.g., single wavelength-based dyessuch as DIBAC, DISBAC (Molecular Devices), and Di-4-ANEPPS (Biotium), ordual wavelength FRET-based dyes such as DiSBAC2, DISBAC3, and CC-2-DMPE(Aurora Biosciences). [Chemical Names—Di-4-ANEPPS (Pyridinium,4-(2-(6-(dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-,hydroxide, Inner salt), DISBAC4(2) (bis-(1,2-dibarbituricacid)-trimethine oxanol), DiSBAC4(3) (bis-(1,3-dibarbituricacid)-trimethine oxanol), CC-2-DMPE (Pacific Blue™1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine, triethylammoniumsalt) and SBFI-AM (1,3-Benzenedicarboxylic acid,4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-,tetrakis[(acetyloxy)methyl] ester].

In one embodiment, the dye-loaded cells are preferably contacted with aknown ENaC inhibitor, e.g., Phenamil, then contacted with test compounds(or controls), and the cell cultures are monitored using standardfluorescence analysis instrumentation such as or VIPR or FLIPR®. Theaddition of NaCl or other test compounds which pharmacologically act onENaC elicit a change in membrane potential which is then detected as achange in the resting fluorescence in a standard fluorescence intensityplate reader (e.g., FLIPR) or voltage intensity plate reader (e.g.VIPR). As such, the method of the present invention can be used toidentify salty taste modulating compounds by monitoring the activity ofENaC in the test cells through fluorescence. For instance, a decrease influorescence may indicate a taste (salty) blocker, while an increase influorescence may indicate a taste (salty) enhancer.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting. It is understoodthat various modifications and changes can be made to the hereindisclosed exemplary embodiments without departing from the spirit andscope of the invention.

Example 1

DNA sequences encoding the alpha, beta and gamma subunit of a human ENaCexpressed in human taste cells were cloned from human kidney cells byRT-PCR.

Methods for Cloning Human Epithelium Sodium Channel Subunit DNASequences (ENaCs)

Human ENaC cDNAs for α, β and γ ENaC were amplified from human kidneycDNA (Origene Technologies Inc.) by PCR using the following primerpairs, respectively: 5′ CGC GGA TCC GCC CAT ACC AGG TCT CAT G 3′ and 5′CCG GAA TTC CTG CAC ATC CTT CAA TCT TGC 3′; 5′ CGC GGA TCC AGC AGG TGCCAC TAT GCA C 3′ and CCG CTC GAG GTC TTG GCT GCT CAG TGA G 3′; 5′ CGCGGA TCC CCT CAA AGT CCC ATC CTC G 3′ and 5′ CCG GAA TTC GAC TAG ATC TGTCTT CTC AAC 3′. The primers were designed to be complementary to 5′ and3′-untranslated region sequence in order to retain the endogenoustranslation initiation signal, and they introduced terminal restrictionendonuclease sites that were used to clone amplified ENaC cDNAs into themammalian expression vector pcDNA3 (Invitrogen) for functionalexpression experiments. The cloned ENaC cDNAs were sequenced andcompared to ENaC sequences in public DNA databanks. Each cloned subunitis a composite of polymorphisms present in different databank alleles;that is, every polymorphism in each cloned subunit identified bypairwise comparison of the cloned subunit to a databank allele could befound in another databank allele. In addition, polymorphisms in clonedENaC subunits were verified by sequencing of cloned cDNAs amplified inindependent PCR experiments.

The nucleic acid sequences encoding cloned sequences alpha, beta andgamma hENaC subunits are respectively contained in SEQ ID NO: 1, SEQ IDNO: 2 and SEQ ID NO: 3 and the corresponding amino acid sequences in SEQID NO: 4, 5 and 6. Each of these DNA sequences was inserted into theexpression vector pcDNA3 to produce alpha, beta and gamma subunitplasmids that express human ENaC subunit polypeptides. Also, the nucleicacid sequence for the human amiloride sensitive sodium channel deltasubunit (ΔNaCh) is contained in SEQ ID NO: 7, which functionsequivalently to the ENaC alpha subunit. The amino acid sequence for thedelta subunit is contained in SEQ ID NO: 8. HEK293T cells weretransiently transfected via Ca²⁺ phosphate with 1:1:1 weight ratios ofα, β, and γ subunit plasmids expressing human ENaC. Such transfectionresulted in a Na⁺ dependent amiloride sensitive fluorescence change, ascompared to mock-transfected cells. With reference to FIG. 1, samples A,B, C, and D were transfected with 1:1:1: ratios of α, β, and γ subunitplasmids at absolute levels of 4, 4, 1, and 0.25 micrograms,respectively. Samples E and F were mock transfected with Beta-gal andpUC DNAs. Transfection efficiency was approximately 40% and cell densitywas approximated 70%. Cells were analyzed using a FLIPR I (MolecularDevices) Instrument using a membrane-potential fluorescent dye. Alltraces shown are from a single plate with A (n=4), B, C, D, E, (n=12),and F (n=8).

As depicted in FIGS. 1, 2, and 3, sodium-dependant amiloride-sensitivechanges in resting potential (hENaC responses) were not significantlyaffected in untransfected HEK293T cells. Further, such resting potentialchanges were greatly enhanced in cells transfected with all threesubunits of the hENaC compared to cells transfected with only the alphasubunit of hENaC (data not shown). Moreover, the ability of NaCl toinduce membrane potential changes, and the effect of amiloride to blockhENaC channel activity follow dose response-relationships similar tothat reported in the literature using low throughputelectrophysiological recording.

Example 2

DNA sequences encoding the alpha, beta and gamma subunits of a humanENaC, SEQID 1, 2, and 3, respectively, were each cloned into theexpression vector pcDNA3 to produce alpha, beta and gamma subunitplasmids that express human ENaC subunit polypeptides. HEK293T cellswere transiently transfected via lipofection with 1:1:1 weight ratios ofα, β, and γ subunit plasmids expressing human ENaC (2 μg of eachsubunit/20 million cells). Transfected cells were plated into 384-wellplates and analyzed on a VIPRII Instrument (Aurora Biosciences) usingvoltage-sensitive fluorescent dyes. Cells expressing ENaC exhibited aNa⁺ dependent fluorescence change, as compared to mock-transfected cells(FIG. 1). In FIG. 2, the Na⁺-dependent fluorescence change is totallyabolished by Phenamil, a known ENaC antagonist. Conversely, anothercompound was found to increase the Na⁺⁻ dependent fluorescence changebut this effect is abolished by Phenamil. This compound is believed tobe an ENaC enhancer, as it acts opposite to Phenamil in this assay forENaC function.

Methods and Materials for Example 2:

-   -   1. All materials used are identified below in the “Materials        Section”.    -   2. HEK293T cells are grown to 80% confluence and dissociated        from the culture dishes with an enzymatic solution        (Trypsin/EDTA) for 3 minutes at 37 C. Detached cells are        analyzed for density and viability using a bench top flow        cytometer (Guava; Guava Technologies). Cells with less than 85%        viability are discarded from the experiment.

[The procedures herein are conditions for transfection of HEK293T cellsequivalent to ten screening 384-well plates (200,000,000 cells). Theseconditions can be altered e.g., by increasing or decreasing cellconfluency by use of different size multi-well plates etc.]

-   -   3. Dissociated cells are washed and recovered in their culture        medium (complete) at a density of ˜1,000,000 cells/ml. Mammalian        expression plasmid DNAs encoding the human ENaC subunits are        mixed in an eppendorf in an equal ratio (10 ug α; 10 ug β and 10        ug γ/20,000,000 cells). 170 μg of carrier plasmid DNA (pUC-18)        is then added to the DNA mix (for a total of 200 μg        DNA/200,000,000 cells). 557 ul of the transfection reagent        TransIT (Panvera Corporation) is added to 20 ml of culture        medium exempt of serum and antibiotic. The DNA solution is then        added to the Transit solution and the DNA-lipid solution is        incubated at room temperature. After 60 minutes, the DNA-lipid        complexes are transferred into the cell solution and volume is        adjusted to 320 ml with complete cell culture medium for a final        density of 635.000 viable cells/mi. (As discussed previously,        the alpha subunit DNA may be interchanged with the delta subunit        DNA and used to produce recombinant cells that express a        functional ENaC comprised of the beta, gamma and delta ENaC        subunits.)    -   4. Black 384-well poly-D-lysine clear bottom screening plates        (Becton Dickinson) are coated with 40 μl/well of a Matrigel        solution (20 μg/ml; Collaborative Biomedical Products) for 1        hour at room temperature. Coating solution is removed and plates        are kept at room temperature until cell plating.    -   5. The cell/DNA solution is plated with a Multidrop into 384        well plates at a density of 50,000 cells/well (80 μl/well).    -   6. 27 hours after plating, cells are washed and loaded with the        membrane potential sensitive dyes (CC2-DMPE and DiSBAC2(3)) as        described below.    -   7. Cells are stimulated with 200 μM compounds ([2×]) and read on        line using a Voltage Intensity Plate Reader (VIPRII; Aurora        Biosciences Corporation). Other concentrations of compounds can        be used in the assay. Buffer preparation and plate layout are        described below in the VIPR. The assay is performed at “room        temperature”, typically about 22° C. but can also be performed        at other temperatures by preheating or cooling the cells and        reagents prior to addition of compounds.

Material

-   -   1. HEK 293T cells growing on 150 cm² flask (Becton Dickinson 0.2        um vented Blueplug seal cap) (37° C., 6% CO₂)    -   2. Dulbecco's Modified Eagle Medium (DMEM) (cat #11965-092 Gibco        BRL) (Kept at 4° C.)    -   3. DMEM with HEPES (DMEMH) (cat #12430-054, Gibco BRL) (Kept at        4° C.)    -   4. Foetal Bovine serum (FBS) (cat#10082-147, Gibco BRL) (Kept in        −20° C.)    -   5. Trypsin EDTA (1×) (cat#25200-072 Gibco-BRL) (Kept in −20° C.)    -   6. TransIT-293 (cat#MIR2705, Panvera) (Kept in 4° C.)    -   7. α, β, and γ ENaC DNA preparations (1 μg/μL each) (Kept in 4°        C.)    -   8. pUC18 carrier DNA ((1 μg/μL) (Kept in 4° C.)    -   9. Matrigel (cat #40230, Collaborative Biomedical Products)

2. Cell Loading

HBSS—Hank's Buffered Saline Solution

DiSBAC₂(3) 5 mM in 100% DMSO 2.6 μM

ESS-CY4 or VABSC-1 200 mM in dH₂0 350 μM

VIPR NMDG BUFFER—see formula in “VIPR Plate Layout” section below:

To Make Volume Components 10 ml 50 ml 100 ml 200 CC2-DMPE(μ) 20 100 200400 Pluronic(μ) 20 100 200 400 HBSS(ml) 10 50 100 200 DiSBAC₂(3)(μ) 5 2550 100 ESS(μ) 17.5 87.5 175 350 VIPR NMDG Buffer (ml) 10 50 100 200

Preparation of CC2-DMPE Loading Buffer

-   -   1. Mix equal volumes of the CC2-DMPE stock solution and Pluronic        F127.    -   2. Add the CC2-DMPE/Pluronic mix to HBSS while vortexing.

Loading of cells with CC2-DMPE

-   -   1. Remove cells from CO₂ incubator.    -   2, Look for variation of density/well    -   3. Prime EMBLA with HBSS    -   4. Wash cells with H BSS 3×80 ul to remove residual growth        medium and serum    -   5. Add 40 μL of 10 μM CC2-DMPE loading buffer to each well    -   6, Look for variation of density/well    -   7. Incubate for 30 minutes at room temperature in the dark.

Preparation of DiSBAC2(3) Loading Buffer

(Can be Done During CC2 Incubation)

-   -   1. Mix DiSBAC₂(3) and ESS-CY4 or VABSC-1, plus double volume of        PluronicF127 of DiSBAC2(3)    -   2. Add the above mix to VIPR NMDG BUFFER, vortex

Loading of Cells with DiSBAC2(3) Loading Buffer

-   -   1. Prime EMBLA with NMDG buffer    -   2. Wash CC2-DMPE-loaded cells using VIPR NMDG buffer as the wash        buffer, 3×80μ/well    -   3. Add 40μ of 2.5 μM DiSBAC2(3), 350 μM ESS-CY4 or VABSC-1        loading buffer to each well    -   4, Look for variation of density/well    -   5. Incubate for 20 minutes at room temperature in the dark        before running on VIPR II

VIPR Plate Layout

NMDG Buffer: NaCl Buffer: ENaC VIPR compound plate preparation 384 wellformat 13 mM NMDG 150 mM NaCl High K Buffer: “VIPR NONE BUFFER” Prepareenough of the following buffers to load all plates:  2 mM KCl  2 mM KCl180 mM KCl  2 mMKCl  2 mMCaCl2  2 mM CaCl2  2 mM CaCl2  2 mMCaCl2•2H2OStock Solutions: Final  1 mM MgCl2  1 mM MgCl2  1 mM MgCl2  1mMMgCl2•6H20 10 mM Hepes 10 mM Hepes  10 mM Hepes 10 mMHEPES acidPluronic F127 100 mg/ml in 100% DMSO  5 mM D-glucose  5 mM D-glucose  5mM D-glucose  5 mMD-glucose DiSBAC₂(3)  5 mM in 100% DMSO  25 μM pH 7.3w/ Tribase ESS-CY4 or VABSC-1 200 mM in dH₂0 350 μM VIPR NMDG BUFFERVIPR Na+ BUFFER VIPR High K buffer Final Volume 10 mls 15 mls 20 mls 100mls 200 mls 400 mls 500 mls DiSBAC₂(3) (μl) 5 7.5 10 50 100 200 250 ESSor VABSL-1 (μL) 17.5 26.26 35 175 350 700 875 Pluronic F127 10 15 20 100300 400 500 Preparation of Column 1 top half Buffer 1. Mix DiSBAC2(3)and ESS-CY4 or VABSC-1 and Pluronic F127 into VIPR NMDG buffer whilevortex Preparation of Column 1 battom half Buffer 1. Mix DiSBAC2(3) andESS-CY4 or VABSC-1 and Pluronic F127 into VIPR HighK buffer while vortexPreparation of Column 24 buffer Add VIPR NMDG buffer into Column 24Preparation of Column 2, 23 70 mM NaCl loading buffer 70 mM NaCl NaClBuffer NMDG Buffer 1. Mix VIPR Na+ buffer with VIPR NMDG buffer to make70 mM NaCl  30 mls 14 16 2. Mix DiSBAC2(3) and ESS-CY4 or VABSC-1 andPluronic F127 into the above buffer while vortex 300 mls 140 180 400 mls214.8 245.3 Adding 2 μl of 100% DMSO into Column1, 2 and 24 Adding 2 μlof 10 nM compound X in 100% DMSO to Column23 Adding all the othercompound into Column3, 22. Plate layout 1 2 3, 4 5, 6 7, 8 9, 10 11, 1213, 14 15, 16 17, 18 19, 20 21, 22 23 24 A NMDG 70 mMNaCl 70 mMNaCl +all 70 mM Na + 200 μM X NMDG the compounds B NMDG 70 mMNaCl 70 mM Na +200 μM X NMDG C NMDG 70 mMNaCl 70 mM Na + 200 μM X NMDG D NMDG 70 mMNaCl70 mM Na + 200 μM X NMDG E NMDG 70 mMNaCl 70 mM Na + 200 μM X NMDG FNMDG 70 mMNaCl 70 mM Na + 200 μM X NMDG G NMDG 70 mMNaCl 70 mM Na + 200μM X NMDG H NMDG 70 mMNaCl 70 mM Na + 200 μM X NMDG I HighK 70 mMNaCl 70mM Na + 200 μM X NMDG J HighK 70 mMNaCl 70 mM Na + 200 μM X NMDG K HighK70 mMNaCl 70 mM Na + 200 μM X NMDG L HighK 70 mMNaCl 70 mM Na + 200 μM XNMDG M HighK 70 mMNaCl 70 mM Na + 200 μM X NMDG N HighK 70 mMNaCl 70 mMNa + 200 μM X NMDG O HighK 70 mMNaCl 70 mM Na + 200 μM X NMDG P HighK 70mMNaCl 70 mM Na + 200 μM X NMDG

Example 3 Improved ENaC Assay Using ENaC Inhibitor (Phenamil)

Modulation of ENaC functions is monitored in human embryonic kidney(HEK)293 cells expressing the three different ENaC subunits. HEK293cells are transiently transfected with the ENaC subunit plasmids usinglipid-based systems. Transfected HEK293 cells are seeded into 384-wellscreening plates and functional expression of ENaC is allowed to proceedfor a total of 24 hours. Cells are then labeled with specific dyes (suchas DisBac2(3) and CC2-DMPE, Panvera) allowing the detection in subtlechanges in membrane potential. Changes in dye fluorescence properties,upon modulation of ENaC functions, are monitored using aVoltage-Intensity-Plate-Reader (VIPR. Panvera). Using this technology,we can detect inhibition of ENaC function (Na+-induced change inmembrane potential dye fluorescence) with increasing concentration of aknown ENaC inhibitor, Phenamil (FIG. 6, blue trace). On the other handan ENaC enhancer (ID #478354) increases ENaC activity by roughly 18%(FIG. 6, difference between red and blue data points in the blackrectangle). Notably, the effect of compound 478354 is greatly improvedby increasing concentrations of Phenamil (FIG. 6, difference between thered and blue data points in yellow rectangle). Under these conditions,478354 increases ENaC activity by as much as 50-60% in the presence of0.2 to 0.5M Phenamil. We conducted more than 241 and 835 independentexperiments in the absence and presence of 0.5 μM Phenamil respectively(FIGS. 7A and 7B). During these experiments, we determined the effect of478354 on the assay window using a Z′ factor. Z′ is a statisticalparameter used to judge the quality of a signal window by quantifyingthe separation of high and low controls sets with respect to theirvariance. A Z′≥0 and ≤1 indicates a meaningful signal window by whichtested chemistries can be compared during screening. In the absence ofPhenamil, a signal window determined by the 478354 high control standardfailed in more than 90% of experiments (FIG. 2A). However, in thepresence of Phenamil, 478354 significantly increased the signal windowin 85% of experiments, with most of the Z′ centered around 0.26 (FIG.2B).

These results indicate that the use of an ENaC inhibitor such asPhenamil, prior to screening with one or more potential ENaC modulators,e.g., ENaC enhancers, enhances signal intensity, thereby significantlyimproving the likelihood of identifying molecules enhancing ENaCactivity in throughput cell-based assays.

Example 4 Electrophysiological Assay for Identifying ENaC ModulatorsUsing Amphibian Oocytes that Express Functional Human ENaC

The oocyte expression system has intrinsic advantages (expressionlevels, robust, low endogenous ion channel expression, et al.) thatrender it useful to examine the effects of compounds on sodium transportthrough ENaC channels. These compounds are candidates for enhancing salttaste perception. The oocyte expression system has been used earlier forthe rapid and robust expression of ion channels, including ENaC, infunctional studies (Dascal. CRC Crit. Rev. Biochem. (1987) 22(4):317-387; Wagner, et al, Cellular Physiology and Biochemistry (2000) 10:1-12; Canessa, et al, Nature (1994) 367: 463-467). Therefore, thissystem was selected for use in a two-electrode voltage clamp assay usingmethods and materials as described below.

The oocyte expression system is comprised of the following steps andmethodologies, which collectively comprise the screen for ENaCenhancers: frog surgery, and oocyte isolation, cRNA preparation, oocytemicroinjection, and measurement of ENaC currents in oocytes usingtwo-electrode voltage clamp electrophysiological recordings. Thefollowing references describe general practices for frog surgery andoocyte isolation (Marcus-Sekura, et al, Methods in Enzymology (1987)152: 284-288; Goldin, Methods in Enzymology (1992) 207: 266-279), cRNApreparation (Swanson, et al, Methods in Enzymology (1992) 207: 310-319;Goldin, et al, Methods in Enzymology (1992) 207: 279-297), oocytemicroinjection (Matten, et al, Methods in Enzymology (1995) 254:458-466; Hitchcock, et al, Methods in Enzymology (1987) 152: 276-284),and two-electrode voltage clamp electrophysiological recording (Stuhmer,Methods in Enzymology (1992) 207: 319-339; Wagner, et al, CellularPhysiology and Biochemistry (2000) 10: 1-12). Each of thesemethodologies, as they pertain to the screen for ENaC enhancers, isdescribed in further detail below.

Frog Surgery and Oocyte Isolation

Female Xenopus laevis South African clawed frogs greater than or equalto 9 cm in length are obtained from NASCO (Fort Atkinson, Wis.). Frogsare anesthetized in 0.15% MS-222 (tricaine or ethyl-3-aminobenzoatemethanesulfonate; Sigma) in distilled water and placed on ice. Usingsterile surgical tools, sequential 1-2 cm incisions are made in theabdomen through both the outer skin layer and the inner peritoneallayer. Excised ovarian lobes (containing 1000-2000 oocytes) are placedin OR-2 calcium-free media (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mMHEPES pH 7.5 with NaOH) and sequentially digested with 2 mg/mlcollagenase type IA (Sigma), prepared immediately before use, for 45 minfollowed by 1 mg/ml collagenase type IA for 15 min on a rocking platformat room temperature. After enzymatic digestion, at which point themajority of oocytes are released from the ovarian lobes, oocytes arerinsed in OR-2 without collagenase and transferred to a Petri dishcontaining Barth's saline (88 mM NaCl, 2 mM KCl, 0.82 mM MgSO₄, 0.33 mMCa(NO₃)2, 0.41 mM CaCl₂, 2.4 mM NaHCO₃, and 5 mM HEPES pH7.5; SpecialtyMedia) supplemented with 2.5 mM sodium pyruvate. Mature stage V or VIoocytes (˜1 mm diameter) containing distinct animal poles, correspondingto the dark side of the egg containing melanin pigment granules, andvegetal poles, corresponding to the light side of the egg containingyolk proteins, are selected for microinjection Frogs are sutured using aC6 needle with a 3-0 black braid suture (Harvard Apparatus) and reusedfor oocyte isolation following a 2-3 month recovery period.

cRNA Preparation

ENaC cRNA is generated using the mMESSAGE mMACHINE kit according to themanufacturer's instructions (Ambion) from α, β, and γ human ENaC DNAplasmids described in our previous patent (PCT WO 02/087306 A2) using T7RNA polymerase to transcribe cRNA in vitro from DNA linearized with EcoRI for α and γ ENaC and linearized with Xho I for β ENaC. cRNA qualityis checked by denaturing agarose gel electrophoresis andspectrophometric absorbance readings at 260 and 280 nm to ensure thatfull-length, non-degraded cRNA is generated.

Microinjection

Microinjection needles are pulled on a Model P-97 Flaming/BrownMicropipette Puller (Sutter Instrument Co.) using borosilicate glasscapillaries (World Precision Instruments Inc.), back-filled with mineraloil (Sigma), and then front-filled with ENaC cRNA using a Nanoliter 2000injector with a Micro4 MicroSyringe Pump Controller (World PrecisionInstruments). Oocytes are microinjected in the animal pole with 10-15 nlcontaining 1-3 ng of each α, β, and γ human ENaC cRNA. Followingmicroinjection, oocytes are transferred to glass scintillation vialscontaining Barth's solution supplemented with 2.5 mM sodium pyruvate andincubated at 18-19° C. overnight under normal atmospheric conditions.During this time, the oocytes translate injected ENaC cRNA into protein.

Measurement of ENaC Currents in Oocytes Using Two-Electrode VoltageClamp Electrophysiological Recordings

Twenty-four hours post-microinjection, ENaC function is measured inoocytes using the two-electrode voltage clamp technique on an OpusXpress6000A parallel oocyte voltage clamp system (Axon Instruments). Thetwo-electrode voltage clamp technique is an electrophysiology methodthat measures the macroscopic electrical current flowing across theentire oocyte membrane though protein channels such as ENaC (Stuhmer,Methods in Enzymology (1992) 207: 319-339). Oocytes are impaled with avoltage-sensing electrode and a current-sensing electrode; the voltage,or potential difference across the oocyte membrane, is clamped to aparticular value using the voltage-sensing electrode and the current, orthe flow of ions across the oocyte membrane, required to maintain thatvoltage is measured using the current-sensing electrode. The OpusXpresssystem is a semi-automated two-electrode voltage clamp workstation thatallows recordings to be made from 8 oocytes simultaneously. Oocyteimpalement is automated and compound delivery is performed bycomputer-controlled fluid handlers from 96-well compound plates. Thismedium-throughput system dramatically increases the number of compoundswe can examine from ˜1 compound per week using a conventional singleoocyte voltage clamp system to ˜60 compounds per week using theOpusXpress system.

Oocytes are placed in the OpusXpress system and bathed in ND-96 solution(96 mM NaCl, 2.5 mM·KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES pH 7.5with NaOH). Oocytes are impaled with voltage-sensing and current-sensingelectrodes, pulled on a Model P-97. Flaming/Brown Micropipette Puller(Sutter Instrument Co.) using borosilicate glass capillaries (WorldPrecision Instruments Inc.) and back-filled with 3M KCl, containingsilver chloride wires. Electrodes exhibit resistances between 2-10 Mohmfor voltage-sensing electrodes and between 0.5-2 Mohm forcurrent-sensing electrodes. Following impalement, oocytes are voltageclamped to ˜60 mV and experimental recordings are initiated. Data areacquired at 50 Hz and low-pass filtered at 5 Hz using a 4-pole Besselfilter.

A preferred procedure used to screen a compound for ENaC enhancement inan oocyte assay according to the invention is as follows (FIG. 8).First, amiloride is applied (1 uM, Sigma). Amiloride is an inhibitorthat blocks sodium transport through ENaC channels and is used as aninternal control to verify that the oocytes express functional ENaCprotein (Canessa, et al, Nature (1994) 367: 463-467). Second, in oocytesexhibiting amiloride inhibition, evidenced by a decrease in currentflowing across the oocyte membrane following amiloride treatment, acompound is applied (concentration between ˜0.1 uM and ˜100 uM). If thecompound functions as an ENaC enhancer, the current passing through ENaCchannels in the oocyte membrane increases. To quantitate the effect of acompound on ENaC function, we use the following formula:[(A−Ao)/(B−Bo)]×−100, where A is the current following compoundtreatment, Ao is the current preceeding compound treatment, B is thecurrent following amiloride treatment, and Bo is the current preceedingamiloride treatment (FIG. 8). This value leads to a % enhancement factorthat is used to gauge the activity of compounds in our assay. Forexample, if the % enhancement factor is equal to 100%, then the compoundincreases ENaC activity 100% over basal control values (in the absenceof compound). % enhancement factors are calculated individually for eachof the oocytes in the OpusXpress system and then an average and standarddeviation are determined for each compound (FIG. 9).

Negative control experiments are performed in oocytes not injected withENaC cRNA to demonstrate that effects observed with compounds in ENaCexpressing oocytes are due to currents flowing through ENaC channels andnot due currents flowing through channels endogenously expressed in theoocyte membrane. (Dascal, CRC Crit. Rev. Biochem. (1987) 22(4):317-387). Compounds specifically enhancing ENaC should not affectcurrents in uninjected oocytes and should exhibit % enhancement factorsof 0 (FIG. 10).

More complex analyses are performed on compounds displaying large %enhancement factors and having no effect on oocytes not injected withENaC cRNA. The assays include current/voltage (I/V) curves, amiloridecompetition experiments, and dose-response curves. For I/V curves,currents are measured in voltage steps from −100 to +60 mV, in 20 mVincrements, in the presence and absence of amiloride, to verify ENaCexpression as above, and in the presence and absence of compound, toinvestigate the magnitude of compound enhancement (FIG. 11). Allnon-ENaC currents (defined as currents not blocked by amiloride) aresubtracted and I/V curves are plotted. The slope of the IN curve isindicative of the magnitude of current enhancement by the compound ofinterest. Strong enhancers exhibit I/V curves with a large positiveslope. In addition, the x-intercept of the IN curve is indicative ofwhat type of ion is being transported in two-electrode voltage clampexperiments. For sodium ion transport, the x-intercept falls within therange of 10-40 mV, depending on the degree of sodium loading in theoocytes. In oocytes not injected with ENaC cRNA, I/V curves performed inthe presence of compound should be identical and superimposible with I/Vcurves performed in the absence of compound (FIG. 11).

Amiloride competition experiments are desirably performed to demonstratethat compound effects are ENaC dependent (FIG. 12). First, amiloride isapplied as above to demonstrate ENaC expression in the oocytes. Then,compound is applied to determine the % enhancement factor. Finally,amiloride and compound are co-applied. For an enhancer to work directlyon the ENaC channel, co-application of amiloride plus compound shouldexhibit an amiloride phenotype, meaning that currents are inhibited andnot enhanced. This assay shows that when the ENaC channel is closed, dueto amiloride, the compound cannot have an enhancing effect; therefore,the compound must directly modulate ENaC channel function.

Dose-response curves are performed to determine the concentration atwhich the compound exhibits half-maximal activity (EC50) (FIG. 13). Thelower the EC50, the more active the compound is as an ENaC enhancer.Dose-response curves are performed by sequentially applying increasingconcentrations of enhancer starting from low doses (˜1 nM) andprogressing to high doses (˜1 mM). % enhancement factors are calculatedas described above and plotted as a function of compound concentrationon a logarithmic scale to determine an EC50 value for the compound.

A flowchart which schematically illustrates the sequence of experimentsperformed to examine the effect of a compound on ENaC function isdepicted in FIG. 14, including screening at a holding potential of −60mV, I/V curves, amiloride competition tests, dose-response curves, andtesting uninjected oocytes.

Analysis of Results

In our previous patent application (PCT WO 02/087306 A2), we utilized ahigh-throughput mammalian cell-based assay in HEK293T human embryonickidney cells that indirectly measured ENaC activity by assaying membranevoltage in ENaC-transfected cells loaded with voltage-sensitivefluorescent probes. Although this approach was high-throughput, andidentified some compounds that modulated ENaC, unfortunately, it was notspecific, and ˜90% of identified compounds did not directly modulateENaC function but instead likely modulated the activity of other ionchannels endogenously expressed in HEK293T cells. The efficacy of suchhigh throughput assays is improved herein by the use of phenamil andsimilar ENaC inhibitors as described supra. In addition, the subjectapplication also provides a more direct (specific), but lower throughputassay methodology to measure ENaC sodium channel function in oocytesusing the two-electrode voltage clamp technique. This system allowsrapid and robust expression of ion channels (˜1 million ENaC channelscan be expressed in the oocyte membrane after only about 18-24 hours).Other advantages of the oocyte expression system include: oocytes arelarge (˜1 mm in diameter) and robust making them easy to handle and workwith; multiple and repetitive recordings can be obtained from the sameoocyte to the same or different compounds of interest; oocytes expressfew endogenous channels at levels sufficient to cause high backgroundcurrent in comparison to the current stemming from an exogenouslyexpressed ion channel; and oocytes allow direct measurement of ionchannel function. Thus, in contrast to assays that indirectly measureENaC function in HEK293T cells using voltage-sensitive probes, theoocyte expression system allows direct measurement of ENaC sodiumchannel current with virtually no background.

Nucleic Acid and Amino Acid Sequences of ENaC Subunits Used in Examples

Length 2010 nucleotides DNAHuman hENaC alpha clone #3-1-1 coding sequence SEQ ID NO: 1atggaggggaacaagctggaggagcaggactctagccctccacagtccactccagggctcatgaaggggaacaagcgtgaggagcaggggctgggccccgaacctgcggcgccccagcagcccacggcggaggaggaggccctgatcgagttccaccgctcctaccgagagctcttcgagttcttctgcaacaacaccaccatccacggcgccatccgcctggtgtgctcccagcacaaccgcatgaagacggccttctgggcagtgctgtggctctgcacctttggcatgatgtactggcaattcggcctgcttttcggagagtacttcagctaccccgtcagcctcaacatcaacctcaactcggacaagctcgtcttccccgcagtgaccatctgcaccctcaatccctacaggtacccggaaattaaagaggagctggaggagctggaccgcatcacagagcagacgctctttgacctgtacaaatacagctccttcaccactctcgtggccggctcccgcagccgtcgcgacctgcgggggactctgccgcaccccttgcagcgcctgagggtcccgcccccgcctcacggggcccgtcgagcccgtagcgtggcctccagcttgcgggacaacaacccccaggtggactggaaggactggaagatcggcttccagctgtgcaaccagaacaaatcggactgcttctaccagacatactcatcaggggtggatgcggtgagggagtggtaccgcttccactacatcaacatcctgtcgaggctgccagagactctgccatccctggaggaggacacgctgggcaacttcatcttcgcctgccgcttcaaccaggtctcctgcaaccaggcgaattactctcacttccaccacccgatgtatggaaactgctatactttcaatgacaagaacaactccaacctctggatgtcttccatgcctggaatcaacaacggtctgtccctgatgctgcgcgcagagcagaatgacttcattcccctgctgtccacagtgactggggcccgggtaatggtgcacgggcaggatgaacctgcctttatggatgatggtggctttaacttgcggcctggcgtggagacctccatcagcatgaggaaggaaaccctggacagacttgggggcgattatggcgactgcaccaagaatggcagtgatgttcctgttgagaacctttacccttcaaagtacacacagcaggtgtgtattcactcctgcttccaggagagcatgatcaaggagtgtggctgtgcctacatcttctatccgcggccccagaacgtggagtactgtgactacagaaagcacagttcctgggggtactgctactataagctccaggttgacttctcctcagaccacctgggctgtttcaccaagtgccggaagccatgcagcgtgaccagctaccagctctctgctggttactcacgatggccctcggtgacatcccaggaatgggtcttccagatgctatcgcgacagaacaattacaccgtcaacaacaagagaaatggagtggccaaagtcaacatcttcttcaaggagctgaactacaaaaccaattctgagtctccctctgtcacgatggtcaccctcctgtccaacctgggcagccagtggagcctgtggttcggctcctcggtgttgtctgtggtggagatggctgagctcgtctttgacctgctggtcatcatgttcctcatgctgctccgaaggttccgaagccgatactggtctccaggccgagggggcaggggtgctcaggaggtagcctccaccctggcatcctcccctccttcccacttctgcccccaccccatgtctctgtccttgtcccagccaggccctgctccctctccagccttgacagcccctccccctgcctatgccaccctgggcccccgcccatctccagggggctctgcaggggccagttcctccacctgtcctctgggggggccctga Length 1923 nucleotides DNAHuman hENaC beta clone #5 coding sequence SEQ ID NO: 2atgcacgtgaagaagtacctGctgaagggcctgcatcggctgcagaagggccccggctacacgtacaaggagctgctggtgtggtactgcgacaacaccaacacccacggccccaagcgcatcatctgtgaggggcccaagaagaaagccatgtggttcctgctcaccctgctcttcgccgccctcgtctgctggcagtggggcatcttcatcaggacctacttgagctgggaggtcagcgtctccctctccgtaggcttcaagaccatggacttccccgccgtcaccatctgcaatgctagccccttcaagtattccaaaatcaagcatttgctgaaggacctggatgagctgatggaagctgtcctggagagaatcctggctcctgagctaagccatgccaatgccaccaggaacctgaacttctccatctggaaccacacacccctggtccttattgatgaacggaacccccaccaccccatggtccttgatctctttggagacaaccacaatggcttaacaagcagctcagcatcagaaaagatctgtaatgcccacgggtgcaaaatggccatgagactatgtagcctcaacaggacccagtgtaccttccggaacttcaccagtgctacccaggcattgacagagtggtacatcctgcaggccaccaacatctttgcacaggtgccacagcaggagctagtagagatgagctaccccggcgagcagatgatcctggcctgcctattcggagctgagccctgcaactaccggaacttcacgtccatcttctaccctcactatggcaactgttacatcttcaactggggcatgacagagaaggcacttccttcggccaaccctggaactgaattcggcctgaagttgatcctggacataggccaggaagactacgtccccttccttgcgtccacggccggggtcaggctgatgcttcacgagcagaggtcataccccttcatcagagatgagggcatctacGccatgtcggggacagagacgtccatcggggtactcgtggacaagcttcagcgcatgggggagccctacagcccgtgcaccgtgaatggttctgaggtccccgtccaaaacttctacagtgactacaacacgacctactccatccaggcctgtcttcgctcctgcttccaagaccacatgatccgtaactgcaactgtggccactacctgtacccactGccccgtggggagaaatactgcaacaaccgggacttcccagactgggcccattgctactcagatctacagatgagcgtggcgcagagagagacctgcattggcatgtgcaaggagtcctgcaatgacacccagtacaagatgaccatctccatggctgactggccttctgaggcctccgaggactggattttccacgtcttgtctcaggagcgggaccaaagcaccaatatcaccctgagcaggaagggaattgtcaagctcaacatctActtccaagaatttaactatcgcaccattgaagaatcagcagccaataacatcgtctggctgctctcgaatctgggtggccagtttggcttctggatggggggctctgtgctgtgcctcatcgagtttggggagatcatcatcgactttgtgtggatcaccatcatcaagctggtggccttggccaagagcctacggcagcggcgagcccaagccagCtacgctggcccaccgcccaccgtggccgagctggtggaggcccacaccaactttggcttccagcctgacacggccccccgcagccccaacactgggccctaccccagtgagcaggccctgcccatcccaggcaccccgccccccaactatgactccctgcgtctgcagccgctggacgtcatcgagtctgacagtgagggtgatgccatctaa Length 1950 nucleotides DNAHuman hENaC gamma clone #3 coding sequence SEQ ID NO: 3atggcacccggagagaagatcaaagccaaaatcaagaagaatctgcccgtgacgggccctcaggcgccgaccattaaagagctgatgcggtggtactgcctcaacaccaacacccatggctgtcgccgcatcgtggtgtcccgcggccgtctgcgccgcctcctctggatcgggttcacactgactgccgtggccctcatcctctggcagtgcgccctcctcgtcttctccttctatactgtctcagtttccatcaaagtccacttccggaagctggattttcctgcagtcaccatctgcaacatcaacccctacaagtacagcaccgttcgccaccttctagctgacttggaacaggagaccagagaggccctgaagtccctgtatggctttccagagtcccggaagcgccgagaggcggagtcctggaactccgtctcagagggaaagcagcctagattctcccaccggattccgctgctgatctttgatcaggatgagaagggcaaggccagggacttcttcacagggAggaagcggaaagtcggcggtagcatcattcacaaggcttcaaatgtcatgcacatcgagtccaagcaagtggtgggattccaactgtgctcaaatgacacctccgactgtgccacctacaccttcagctcgggaatcaatgccattcaggagtggtataagctacactacatgaacatcatggcacaggtgcctctggagaagaaaatcaacatgagctattctgctgaggagctgctggtgacctgcttctttgatggagtgtcctgtgatgccaggaatttcacgcttttCcaccacccgatgcatgggaattgctatactttcaacaacagagaaaatgagaccattctcagcacctccatggggggcagcgaatatgggctgcaagtcattttgtacataaacgaagaggaatacaacccattcctcgtgtcctccactggagctaaggtgatcatccatcggcaggatgagtatcccttcgtcgaagatgtgggaacagagattgagacagcaatggtcacctctataggaatgcacctgacagagtccttcaagctgagtgagccctacagtcagtgcacggaggacgggagtgacgtgccaatcaggaacatctacaacgctgcctactcgctccagatctgccttcattcatgcttccagacaaagatggtggagaaatgtgggtgtgcccagtacagccagcctctacctcctgcagccaactactgcaactaccagcagcaccccaactggatgtattgttactaccaactgcatcgagcctttgtccaggaagagctgggctgccagtctgtgtgcaaggaagcctgcagctttaaagagtggacactaaccacaagcctggcacaatggccatctgtggtttcggagaagtggttgctgcctgttctcacttgggaccaaggccggcaagtaaacaaaaagctcaacaagacagacttgGccaaactcttgatattctacaaagacctgaaccagagatccatcatggagagcccagccaacagtattgagatgcttctgtccaacttcggtggccagctgggcctgtggatgagctgctctgttgtctgcgtcatcgagatcatcgaggtcttcttcattgacttcttctctatcattgcccgccgccagtggcagaaagccaaggagtggtgggcctggaaacaggctcccccatgtccagaagctccccgtagcccacagggccaggacaatccagccctggatatagacgatgacctacccactttcaactctgctttgcacctgcctccaGccctaggaacccaagtgcccggcacaccgccccccaaatacaataccttgcgcttggagagggccttttccaaccagctcacagatacccagatgctAgatgagctctga Length 669 amino acids PRTHuman hENaC alpha clone #3-1-1 amino acid sequence SEQ ID NO: 4MEGNKLEEQDSSPPQSTPGLMKGNKREEQGLGPEPAAPQQPTAEEEALIEFHRSYRELFEFFCNNTTIHGAIRLVCSQHNRMKTAFWAVLWLCTFGMMYWQFGLLFGEYFSYPVSLNINLNSDKLVFPAVTICTLNPYRYPEIKEELEELDRITEQTLFDLYKYSSFTTLVAGSRSRRDLRGTLPHPLQRLRVPPPPHGARRARSVASSLRDNNPQVDWKDWKIGFQLCNQNKSDCFYQTYSSGVDAVREWYRFHYINILSRLPETLPSLEEDTLGNFIFACRFNQVSCNQANYSHFHHPMYGNCYTFNDKNNSNLWMSSMPGINNGLSLMLRAEQNDFIPLLSTVTGARVMVHGQDEPAFMDDGGFNLRPGVETSISMRKETLDRLGGDYGDCTKNGSDVPVENLYPSKYTQQVCIHSCFQESMIKECGCAYIFYPRPQNVEYCDYRKHSSWGYCYYKLQVDFSSDHLGCFTKCRKPCSVTSYQLSAGYSRWPSVTSQEWVFQMLSRQNNYTVNNKRNGVAKVNIFFKELNYKTNSESPSVTMVTLLSNLGSQWSLWFGSSVLSVVEMAELVFDLLVIMFLMLLRRFRSRYWSPGRGGRGAQEVASTLASSPPSHFCPHPMSLSLSQPGPAPSPALTAPPPAYATLGPRPSPGGSAGASSSTCPLGGP Length 640 amino acids PRTHuman hENaC beta clone #5 amino acid sequence SEQ ID NO: 5MHVKKYLLKGLHRLQKGPGYTYKELLVWYVDNTNTHGPKRIICEGPKKKAMWFLLTLLFAALVCWQWGIFIRTYLSWEVSVSLSVGFKTMDFPAVTICNASPFKYSKIKHLLKDLDELMEAVLERILAPELSHANATRNLNFSIWNHTPLVLIDERNPHHPMVLDLFGDNHNGLTSSSASEKICNAHGCKMAMRLCSLNRTQCTFRNFTSATQALTEWYILQATNIFAQVPQQELVEMSYPGEQMILACLFGAPECNYRNFTSIFYPHYGNCYIFNWGMTEKALPSANPGTEFGLKLILDIGQEDYVPFLASTAGVRLMLHEQRSYPFIRDEGIYAMSGTETSIGVLVDKLQRMGEPYSPCTVNGSEVPVQNFYSDYNTTYSIQACLRSCFQDHMIRNCNCGHYLYPLPRGEKYCNNRDFPDWAHCYSDLQMSVAQRETCIGMCKESCNDTQYKMTISMADWPSEASEDWIFHVLSQERDQSTNITLSRKGIVKLNIYFQEFNYRTIEESAANNIVWLLSNLGGQFGFWMGGSVLCLIEFGEIIIDFVWITIIKLVALAKSLRQRRAQASYAGPPPTVAELVEAHTNFGFQPDTAPRSPNTGPYPSEQALPIPGTPPPNYDSLRLQPLDVIESDSEGDAILength 650 amino acids PRTHuman hENaC gamma clone #3 amino acid sequence SEQ ID NO: 6MAPGEKIKAKIKKNLPVTGPQAPTIKELMRWYCLNTNTHGCRRIVVSRGRLRRLLWIGFTLTAVALILWQCALLVFSFYTVSVSIKVHFRKLDFPAVTICNINPYKYSTVRHLLADLEQETREALKSLYGFPESRKRREAESWNSVSEGKQPRFSHRIPLLIFDQDEKGKARDFFTGRKRKVGGSIIHKANVMHIESKQVVGFQLCSNDTSDCATYTFSSGINAIQEWYKLHYMNIMAQVPLEKKINMSYSAEELLVTCFFDGVSCDARNFTLFHHPMHGNCYTFNNRENETILSTSMGGSEYGLQVILYINEEEYNPFLVSSTGAKVIIHRQDEYPFVEDVGTEIETAMVTSIGMHLTESFKLSEPYSQCTEDGSDVPIRNIYNAAYSLQICLHSVFQTKMVEKCGCAQYSQPLPPAANYCNYQQHPNWMYCYYQLHRAFVQEELGCQSVCKEACSFKEWTLTTSLAQWPSVVSEKWLLPVLTWDQGRQVNKKLNKTDLAKLLIFYKDLNQRSIMESPANSIEMLLSNFGGQLGLWMNSCSVVCVIEIIEVFFIDFFSIIARRQWQKAKEWWAWKQAPPCPEAPRSPQGQDNPALDIDDDLPTFNSALHLPPALGTQVPGTPPPKYNTLRLERAFSNQLTDTQMLDEL Length 1917 nucleotides DNAgi|1066456|gb|U38254.1|HSU38254 Human amiloride sensitive sodium channel deltasubunit (NaCh) mRNA, complete coding sequence SEQ ID NO: 7ATGGCTGAGCACCGAAGCATGGACGGGAGAATGGAAGCAGCCACACGGGGGGGCTCTCACCTCCAGGCTGCAGCCCAGACGCCCCCCAGGCCGGGGCCACCATCAGCACCACCACCACCACCCAAGGAGGGGCACCAGGAGGGGCTGGTGGAGCTGCCCGCCTCGTTCCGGGAGCTGCTCACCTTCTTCTGCACCAATGCCACCATCCACGGCGCCATCCGCCTGGTCTGCTCCCGCGGGAACCGCCTCAAGACGACGTCCTGGGGGCTGCTGTCCCTGGGAGCCCTGGTCGCGCTCTGCTGGCAGCTGGGGCTCCTCTTTGAGCGTCACTGGCACCGCCCGGTCCTCATGGCCGTCTCTGTGCACTCGGAGCGCAAGCTGCTCCCGCTGGTCACCCTGTGTGACGGGAACCCACGTCGGCCGAGTCCGGTCCTCCGCCATCTGGAGCTGCTGGACGAGTTTGCCAGGGAGAACATTGACTCCCTGTACAACGTCAACCTCAGCAAAGGCAGAGCCGCCCTCTCCGCCACTGTCCCCCGCCACGAGCCCCCCTTCCACCTGGACCGGGAGATCCGTCTGCAGAGGCTGAGCCACTCGGGCAGCCGGGTCAGAGTGGGGTTCAGACTGTGCAACAGCACGGGCGGCGACTGCTTTTACCGAGGCTACACGTCAGGCGTGGCGGCTGTCCAGGACTGGTACCACTTCCACTATGTGGATATCCTGGCCCTGCTGCCCGCGGCATGGGAGGACAGCCACGGGAGCCAGGACGGCCACTTCGTCCTCTCCTGCAGTTACGATGGCCTGGACTGCCAGGCCCGACAGTTCCGGACCTTCCACCACCCCACCTACGGCAGCTGCTACACGGTCGATGGCGTCTGGACAGCTCAGCGCCCCGGCATCACCCACGGAGTCGGCCTGGTCCTCAGGGTTGAGCAGCAGCCTCACCTCCCTCTGCTGTCCACGCTGGCCGGCATCAGGGTCATGGTTCACGGCCGTAACCACACGCCCTTCCTGGGGCACCACAGCTTCAGCGTCCGGCCAGGGACGGAGGCCACCATCAGCATCCGAGAGGACGAGGTGCACCGGCTCGGGAGCCCCTACGGCCACTGCACCGCCGGCGGGGAAGGCGTGGAGGTGGAGCTGCTACACAACACCTCCTACACCAGGCAGGCCTGCCTGGTGTCCTGCTTCCAGCAGCTGATGGTGGAGACCTGCTCCTGTGGCTACTACCTCCACCCTCTGCCGGCGGGGGCTGAGTACTGCAGCTCTGCCCGGCACCCTGCCTGGGGACACTGCTTCTACCGCCTCTACCAGGACCTGGAGACCCACCGGCTCCCCTGTACCTCCCGCTGCCCCAGGCCCTGCAGGGAGTCTGCATTCAAGCTCTCCACTGGGACCTCCAGGTGGCCTTCCGCCAAGTCAGCTGGATGGACTCTGGCCACGCTAGGTGAACAGGGGCTGCCGCATCAGAGCCACAGACAGAGGAGCAGCCTGGCCAAAATCAACATCGTCTACCAGGAGCTCAACTACCGCTCAGTGGAGGAGGCGCCCGTGTACTCGGTGCCGCAGCTGCTCTCCGCCATGGGCAGCCTCTACAGCCTGTGGTTTGGGGCCTCCGTCCTCTCCCTCCTGGAGCTCCTGGAGCTGCTGCTCGATGCTTCTGCCCTCACCCTGGTGCTAGGCGGCCGCCGGCTCCGCAGGGCGTGGTTCTCCTGGCCCAGAGCCAGCCCTGCCTCAGGGGCGTCCAGCTCAAGCCAGAGGCCAGTCAGATGCCCCCGCCTGCAGGCGGCACGTCAGATGACCCGGAGCCCAGCGGGCCTCATCTCCCACGGGTGATGCTTCCAGGGGTTCTGGCGGGAGTCTCAGCCGAAGAGAGCTGGGCTGGGCCCCAGCCCCTTGAGACTCTGGACACCTGALength 638 nucleotides PRTgi|1710872|sp|P51172|SCAD_HUMAN Amiloride-sensitive sodium channel delta-subunitamino acid sequence (Epithelial Na+ channel delta subunit) (Delta ENaC)(Nonvoltage-gated sodium channel 1 delta subunit) (SCNED) (Delta NaCh)SEQ ID NO: 8MAEHRSMDGRMEAATRGGSHLQAAAQTPPRPGPPSAPPPPPKEGHQEGLVELPASFRELLTFFCTNATIHGAIRLVCSRGNRLKTTSWGLLSLGALVALCWQLGLLFERHNWHRPVLMAVSVHSERKLLPLVTLCDGNPRRPSPVLRHLELLDEFARENIDSLYNVNLSKGRAALSATVPRHEPPFHLDREIRLQRLSHSGSRVRVGFRLCNSTGGDCFYRGYTSGVAAVQDWYHFHYVDILALLPAAWEDSHGSQDGHVFLSCSYDGLDCQARQFRTFHHPTYGSCYTVDGVWTAQRPGITHGVGLVLRVEQQPHLPLLSTLAGIRVMVHGRNHTPFLGHHSFSVRPGTEATISIREDEVHRLGSPYGHCTAGGEGVEVELLHNTSYTRQACLVSCFQQLMVETCSCGYYLHPLPAGAEYCSSARHPAWGHCFYRLYQDLETHRLPCTSRCPRPCRESAFKLSTGTSRWPSAKSAGWTLATLGEQGLPHQSHRQRSSLAKINIVYQELNYRSVEEAPVYSVPQLLSAMGSLYSLWFGASVLSLLELLELLLDASALTLVLGGRRLRRAWFSWPRASPASGASSIKPEASQMPPPAGGTSDDPEPSGPHLPRVMLPGVLAGVSAEESWAGPQPLETLDT

While the invention has been described by way of examples and preferredembodiments, it is understood that the words which have been used hereinare words of description, rather than words of limitation. Changes maybe made, within the purview of the appended claims, without departingfrom the scope and spirit of the invention in its broader aspects.Although the invention has been described herein with reference toparticular means, materials, and embodiments, it is understood that theinvention is not limited to the particulars disclosed. The inventionextends to all equivalent structures, means, and uses which are withinthe scope of the appended claims.

1-110. (canceled)
 111. An isolated mammalian cell or cell populationthat expresses a functional hENaC comprising a delta subunit which is atleast 95% identical to the polypeptide in SEQ ID NO:7 and an alpha andbeta subunit which are respectively at least 95% identical to thepolypeptides in SEQ ID NO: 1 and
 2. 112. The isolated cell or cellpopulation of claim 111, which is human.
 113. The isolated cell or cellpopulation of claim 111, which is selected from the group consisting ofMDCK, BHK, COS, NIH3T3, Swiss3T3 and CHO cells.
 114. The isolated cellor cell population of claim 111 wherein said cell or cells transientlyexpress the alpha (or delta), beta and gamma hENaC subunits.
 115. Theisolated cell or cell population of claim 111, wherein said cell orcells stably express the alpha (or delta), beta and gamma hENaCsubunits.
 116. The isolated cell or cell population of claim 111,wherein said cell or cells are comprised in a multi-well test platedevice.
 117. The isolated cell or cell population of claim 111, whereinsaid cell or cells are loaded with a membrane potential dye.
 118. Theisolated cell or cell population of claim 111, wherein said cells aregrown to about 80% confluence.
 119. The isolated cell or cell populationof claim 118, wherein the membrane potential dye is CC2-DMPVE,DiSBAC2(3) or ESS-CY4.
 120. The isolated cell or cell population ofclaim 111, wherein the delta subunit has the sequence in SEQ ID NO:7.121. The isolated cell or cell population of claim 111, wherein thealpha and beta subunits respectively contain the polypeptides in SEQ IDNO: 1 and
 2. 122. A method of identifying a compound which modulatestaste by: (i) contacting a recombinant or isolated eukaryotic cell orcell population that expresses a functional human Epithelial SodiumChannel (hENaC) comprising a delta subunit which is at least 95%identical to the polypeptide encoded by SEQ ID NO:7 and an alpha andbeta subunit which are respectively at least 95% identical to thepolypeptides encoded by SEQ ID NO: 1 and 2 with at least one compound,(ii) identifying whether the compound modulates the activity of saidfunctional hENaC; and (iii) based on whether the compound modulatesactivity of said hENaC identifying the compound as one potentiallymodulating taste.
 123. The method of claim 122, wherein the eukaryoticcell is selected from mammalian cells, yeast, Xenopus oocytes and insectcells.
 124. The method of claim 122, which includes testing theidentified compound in a taste test.
 125. The method of claim 122,wherein the isolated eukaryotic cell or cell population is human. 126.The method of claim 122, wherein the isolated eukaryotic cell or cellpopulation is selected from the group consisting of MDCK, BHK, COS,NIH3T3, Swiss3T3 and CHO cells.
 127. The method of claim 122, whereinthe isolated eukaryotic cell or cell population transiently or stablyexpresses the alpha (or delta), beta and gamma hENaC subunits.
 128. Themethod of claim 122, wherein the isolated eukaryotic cell or cellpopulation are comprised in a multi-well test plate device and theisolated eukaryotic cell or cell population are loaded with a membranepotential dye.
 129. The method of claim 122, wherein the expressed deltasubunit is encoded by the sequence in SEQ ID NO:7 and the expressedalpha and beta subunits respectively contain the polypeptides encoded bySEQ ID NO: 1 and 2.